RESUMO
Abnormal numerical and structural chromosome content is frequently found in human cancer. To test the role of aneuploidy in tumor initiation and progression, we generated mice with random aneuploidies by transient induction of polo-like kinase 4 (Plk4), a master regulator of centrosome number. Short-term chromosome instability (CIN) from transient Plk4 induction resulted in formation of aggressive T-cell lymphomas in mice with heterozygous inactivation of one p53 allele and accelerated tumor development in the absence of p53. Transient CIN increased the frequency of lymphoma-initiating cells with a specific karyotype profile, including trisomy of chromosomes 4, 5, 14, and 15 occurring early in tumorigenesis. Tumor development in mice with chronic CIN induced by an independent mechanism (through inactivation of the spindle assembly checkpoint) gradually trended toward a similar karyotypic profile, as determined by single-cell whole-genome DNA sequencing. Overall, we show how transient CIN generates cells with random aneuploidies from which ones that acquire a karyotype with specific chromosome gains are sufficient to drive cancer formation, and that distinct CIN mechanisms can lead to similar karyotypic cancer-causing outcomes.
Assuntos
Aneuploidia , Instabilidade Cromossômica , Animais , Transformação Celular Neoplásica/genética , Centrossomo , Instabilidade Cromossômica/genética , Evolução Clonal , Instabilidade Genômica/genética , CamundongosRESUMO
Chromosome instability (CIN) and aneuploidy are hallmarks of cancer cells, typically associated with aggressiveness and poor outcomes. Historically, the causative link between aneuploidy and cancer has been difficult to study due to its intrinsic complexity and the poor fitness of aneuploid cells. In this issue of Genes & Development, two companion papers (Trakala and colleagues [pp. 1079-1092] and Shoshani and colleagues [pp. 1093-1108]) exploited sophisticated mouse models to study the progression of aneuploidy from early phases to established tumors. Both groups observed that, while in the early nontumoral cells aneuploidy is characterized by random chromosomal gains, established tumors display a stereotypic karyotype with recurrent gains of only a few chromosomes. Thus, aneuploidy in tumors is not random but shows reproducible patterns of chromosomal changes induced by mechanisms that these two studies are beginning to unveil.
Assuntos
Aneuploidia , Neoplasias , Animais , Instabilidade Cromossômica/genética , Aberrações Cromossômicas , Cariótipo , Camundongos , Neoplasias/genética , Neoplasias/patologiaRESUMO
Centrosomes are the canonical microtubule organizing centers (MTOCs) of most mammalian cells, including spermatocytes. Centrosomes comprise a centriole pair within a structurally ordered and dynamic pericentriolar matrix (PCM). Unlike in mitosis, where centrioles duplicate once per cycle, centrioles undergo two rounds of duplication during spermatogenesis. The first duplication is during early meiotic prophase I, and the second is during interkinesis. Using mouse mutants and chemical inhibition, we have blocked centriole duplication during spermatogenesis and determined that non-centrosomal MTOCs (ncMTOCs) can mediate chromosome segregation. This mechanism is different from the acentriolar MTOCs that form bipolar spindles in oocytes, which require PCM components, including gamma-tubulin and CEP192. From an in-depth analysis, we identified six microtubule-associated proteins, TPX2, KIF11, NuMA, and CAMSAP1-3, that localized to the non-centrosomal MTOC. These factors contribute to a mechanism that ensures bipolar MTOC formation and chromosome segregation during spermatogenesis when centriole duplication fails. However, despite the successful completion of meiosis and round spermatid formation, centriole inheritance and PLK4 function are required for normal spermiogenesis and flagella assembly, which are critical to ensure fertility.
Assuntos
Centríolos , Segregação de Cromossomos , Proteínas Associadas aos Microtúbulos , Centro Organizador dos Microtúbulos , Espermatócitos , Espermatogênese , Centríolos/metabolismo , Centríolos/genética , Animais , Masculino , Camundongos , Espermatogênese/genética , Espermatócitos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Meiose/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMO
Centriole duplication is tightly controlled to maintain correct centriole number through the cell cycle. Key to this is the regulated degradation of PLK4, the master regulator of centriole duplication. Here, we show that the Rac1 guanine nucleotide exchange factor (GEF) Tiam1 localises to centrosomes during S-phase, where it is required for the maintenance of normal centriole number. Depletion of Tiam1 leads to an increase in centrosomal PLK4 and centriole overduplication, whereas overexpression of Tiam1 can restrict centriole overduplication. Ultimately, Tiam1 depletion leads to lagging chromosomes at anaphase and aneuploidy, which are potential drivers of malignant progression. The effects of Tiam1 depletion on centrosomal PLK4 levels and centriole overduplication can be rescued by re-expression of both wild-type Tiam1 and catalytically inactive (GEF*) Tiam1, but not by Tiam1 mutants unable to bind to the F-box protein ßTRCP (also known as F-box/WD repeat-containing protein 1A) implying that Tiam1 regulates PLK4 levels through promoting ßTRCP-mediated degradation independently of Rac1 activation.
Assuntos
Centríolos , Proteínas Serina-Treonina Quinases , Ciclo Celular , Proteínas de Ciclo Celular/genética , CentrossomoRESUMO
Centriole duplication occurs once per cell cycle and is regulated by Polo-like kinase 4 (PLK4). Overexpression of PLK4 in somatic cells can lead to the excessive formation of centrioles, directly causing chromosome segregation errors and tumorigenesis. In this study, we described our efforts to develop a series of PLK4 inhibitors with 1H-pyrazolo[3,4-d]pyrimidine core, and further structure- and receptor-based design and optimization resulted in a potent inhibitor WY29 (IC50 = 0.027 µM), which exhibited good selectivity to other PLK family members (PLK1-3). At the cellular level, compound WY29 showed excellent antiproliferative activity against three breast cancer cell lines (MCF-7, BT474, and MDA-MB-231) while weak inhibitory activity was found on normal cell line HUVECs. In addition, the in vitro preliminary drug-like properties evaluation of compound WY29 showed outstanding stability in human plasma and liver microsomes, and weak inhibitory activity against the major subtypes of human cytochrome P450. Also, the drug-like properties prediction of compound WY29 displayed remarkable drug-like properties (drug-likeness mode score: 1.06). In conclusion, these results support the further development of compound WY29 as a lead compound for PLK4-targeted anticancer drug discovery.
Assuntos
Inibidores de Proteínas Quinases , Pirimidinas , Humanos , Relação Estrutura-Atividade , Linhagem Celular Tumoral , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Proteínas Serina-Treonina QuinasesRESUMO
BACKGROUND: Identification of novel molecular target(s) is important for designing newer mechanistically driven approaches for the treatment of prostate cancer (PCa), which is one of the main causes of morbidity and mortality in men. In this study, we determined the role of polo-like kinase 4 (PLK4), which regulates centriole duplication and centrosome amplification (CA), in PCa. MATERIALS AND METHODS: Employing human PCa tissue microarrays, we assessed the prevalence of CA, correlated with Gleason score, and estimated major causes of CA in PCa (cell doubling vs. centriole overduplication) by staining for mother/mature centrioles. We also assessed PLK4 expression and correlated it with CA in human PCa tissues and cell lines. Further, we determined the effects of PLK4 inhibition in human PCa cells. RESULTS: Compared to benign prostate, human PCa demonstrated significantly higher CA, which was also positively correlated with the Gleason score. Further, most cases of CA were found to arise by centriole overduplication rather than cell doubling events (e.g., cytokinesis failure) in PCa. In addition, PLK4 was overexpressed in human PCa cell lines and tumors. Moreover, PLK4 inhibitors CFI-400945 and centrinone-B inhibited cell growth, viability, and colony formation of both androgen-responsive and androgen-independent PCa cell lines. PLK4 inhibition also induced cell cycle arrest and senescence in human PCa cells. CONCLUSIONS: CA is prevalent in PCa and arises predominantly by centriole overduplication as opposed to cell doubling events. Loss of centrioles is cellular stress that can promote senescence and suggests that PLK4 inhibition may be a viable therapeutic strategy in PCa.
Assuntos
Androgênios , Neoplasias da Próstata , Proteínas Serina-Treonina Quinases , Androgênios/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Centrossomo/metabolismo , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
PLK4 has emerged as a prime target for cancer therapeutics, and its overexpression is frequently observed in various types of human cancer. Recent studies have further revealed an unexpected oncogenic activity of PLK4 in regulating cancer cell migration and invasion. However, the molecular basis behind the role of PLK4 in these processes still remains only partly understood. Our previous work has demonstrated that an intact CEP85-STIL binding interface is necessary for robust PLK4 activation and centriole duplication. Here, we show that CEP85 and STIL are also required for directional cancer cell migration. Mutational and functional analyses reveal that the interactions between CEP85, STIL and PLK4 are essential for effective directional cell motility. Mechanistically, we show that PLK4 can drive the recruitment of CEP85 and STIL to the leading edge of cells to promote protrusive activity, and that downregulation of CEP85 and STIL leads to a reduction in ARP2 (also known as ACTR2) phosphorylation and reorganization of the actin cytoskeleton, which in turn impairs cell migration. Collectively, our studies provide molecular insight into the important role of the CEP85-STIL complex in modulating PLK4-driven cancer cell migration.This article has an associated First Person interview with the first author of the paper.
Assuntos
Centríolos , Proteínas Serina-Treonina Quinases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimento Celular , Centríolos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Centrosome amplification (CA) refers to a numerical increase in centrosomes resulting in cells with more than two centrosomes. CA has been shown to initiate tumorigenesis and increase the invasive potential of cancer cells in genetically modified experimental models. Hexavalent chromium is a recognized carcinogen that causes CA and tumorigenesis as well as promotes cancer metastasis. Thus, CA appears to be a biological link between chromium and cancer. In the present study, we investigated how chromium triggers CA. Our results showed that a subtoxic concentration of chromium-induced CA in HCT116 colon cancer cells, resulted in the production of reactive oxygen species (ROS), activated ATF6 without causing endoplasmic reticulum stress, and upregulated the protein level of PLK4. Inhibition of ROS production, ATF6 activation, or PLK4 upregulation attenuated CA. Inhibition of ROS using N-acetyl-l-cysteine (NAC) inhibited chromium-induced activation of ATF6 and upregulation of PLK4. ATF6-specific siRNA knocked down the protein level and activation of ATF6, and upregulated PLK4, with no effect on ROS production. Knockdown of PLK4 protein had no effect on chromium-induced ROS production or activation of ATF6. In conclusion, our results suggest that hexavalent chromium induces CA via the ROS-ATF6-PLK4 pathway and provides molecular targets for inhibiting chromium-mediated CA, which may be useful for the assessment of CA in chromium-promoted tumorigenesis and cancer cell metastasis.
Assuntos
Neoplasias do Colo , Proteínas Serina-Treonina Quinases , Fator 6 Ativador da Transcrição/metabolismo , Carcinogênese/metabolismo , Transformação Celular Neoplásica/metabolismo , Centrossomo/metabolismo , Cromo/metabolismo , Cromo/toxicidade , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Humanos , Espécies Reativas de Oxigênio/metabolismoRESUMO
The centriole organelle consists of microtubules (MTs) that exhibit a striking 9-fold radial symmetry. Centrioles play fundamental roles across eukaryotes, notably in cell signaling, motility and division. In this Cell Science at a Glance article and accompanying poster, we cover the cellular life cycle of this organelle - from assembly to disappearance - focusing on human centrioles. The journey begins at the end of mitosis when centriole pairs disengage and the newly formed centrioles mature to begin a new duplication cycle. Selection of a single site of procentriole emergence through focusing of polo-like kinase 4 (PLK4) and the resulting assembly of spindle assembly abnormal protein 6 (SAS-6) into a cartwheel element are evoked next. Subsequently, we cover the recruitment of peripheral components that include the pinhead structure, MTs and the MT-connecting A-C linker. The function of centrioles in recruiting pericentriolar material (PCM) and in forming the template of the axoneme are then introduced, followed by a mention of circumstances in which centrioles form de novo or are eliminated.
Assuntos
Centríolos/ultraestrutura , Microtúbulos/ultraestrutura , Biogênese de Organelas , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Embrião de Mamíferos , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Marsileaceae/genética , Marsileaceae/metabolismo , Marsileaceae/ultraestrutura , Camundongos , Microtúbulos/metabolismo , Mitose , Naegleria/genética , Naegleria/metabolismo , Naegleria/ultraestrutura , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisRESUMO
RNA-binding motif protein 10 (RBM10) primarily regulates alternative splicing of certain genes. Loss-of-function mutations in RBM10 have been frequently reported in patients with various cancers. However, how RBM10 levels affect cell proliferation and tumorigenesis remains unknown. To elucidate the role of RBM10 in cell proliferation, we established HepG2-RBM10 knockout cell lines and derivative doxycycline-inducible RBM10-expressing cells. RBM10 over-expression caused growth arrest in the M phase with a monopolar spindle because of impaired centriole duplication. Two RBM10 splicing mutants, one with F345A/F347A and the other with only the C-terminal half (401-930), were sufficient to cause growth arrest, whereas an RBM10 mutant with cytoplasmic localization forced by an NES did not show growth arrest. RBM10 over-expression induced the formation of many large nuclear domains containing RBM10, PLK4, STIL and SAS6, which are the regulatory proteins involved in centriole duplication. Consistently, the centrioles in the RBM10-over-expressing HepG2 cells lost PLK4 and STIL, accounting for the unsuccessful centriole duplication. In contrast, RBM10 depletion resulted in elevated levels of cytoplasmic PLK4 with a concomitant increase in the number of centrioles in HepG2 cells but not in A549 cells. Thus, nuclear RBM10 regulates normal chromosomal division in a cell-type-specific manner, independent of alternative RNA splicing.
Assuntos
Núcleo Celular/metabolismo , Centríolos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Carcinogênese/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Células Hep G2 , Humanos , TranscriptomaRESUMO
Multiple circular RNAs (circRNAs) have been identified to act as essential mediators in diverse human cancers. However, the roles of circRNAs in neuroblastoma (NB) are largely unknown. In this study, we aimed to explore the function of circKIF2A in NB. Quantitative real-time polymerase chain reaction was executed to detect the levels of circKIF2A, KIF2A mRNA, miR-129-5p and polo-like kinase 4 (PLK4) mRNA. Actinomycin D assay and RNase R digestion assay were conducted to analyze the feature of circKIF2A. 3-(4, 5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, transwell assay and specific kits were utilized to evaluate cell proliferation, metastasis and glycolysis, respectively. Western blot assay was performed to examine the protein levels of matrix metalloproteinase 2 (MMP2), MMP9 and PLK4. Bioinformatics analysis, RNA pull-down assay and dual-luciferase reporter assay were conducted to analyze the relationship between miR-129-5p and circKIF2A or PLK4. Murine xenograft model assay was done to investigate the role of circKIF2A in NB in vivo. CircKIF2A level was increased in NB tissue samples and cell lines. Silencing of circKIF2A impeded NB cell proliferation, migration, invasion and glycolysis. For mechanism analysis, circKIF2A could positively modulate PLK4 expression via sponging miR-129-5p. Moreover, miR-129-5p inhibition reversed the inhibitory effects of circKIF2A silencing on the behaviors of NB cells. MiR-129-5p overexpression weakened the malignant biological behaviors of NB cells by targeting PLK4. Additionally, circKIF2A knockdown hampered tumorigenesis in vivo. CircKIF2A knockdown suppressed cell proliferation, migration, invasion and glycolysis via downregulating PLK4 expression through miR-129-5p.
Assuntos
Movimento Celular , Proliferação de Células , Glicólise , MicroRNAs/metabolismo , Proteínas de Neoplasias/metabolismo , Neuroblastoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Circular/metabolismo , RNA Neoplásico/metabolismo , Transdução de Sinais , Células Endoteliais da Veia Umbilical Humana , Humanos , MicroRNAs/genética , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Serina-Treonina Quinases/genética , RNA Circular/genética , RNA Neoplásico/genéticaRESUMO
Mammalian epithelial cells use a pair of parental centrioles and numerous deuterosomes as platforms for efficient basal body production during multiciliogenesis. How deuterosomes form and function, however, remain controversial. They are proposed to arise either spontaneously for massive de novo centriole biogenesis or in a daughter centriole-dependent manner as shuttles to carry away procentrioles assembled at the centriole. Here, we show that both parental centrioles are dispensable for deuterosome formation. In both mouse tracheal epithelial and ependymal cells (mTECs and mEPCs), discrete deuterosomes in the cytoplasm are initially procentriole-free. They emerge at widely dispersed positions in the cytoplasm and then enlarge, concomitant with their increased ability to form procentrioles. More importantly, deuterosomes still form efficiently in mEPCs whose daughter centriole or even both parental centrioles are eliminated through shRNA-mediated depletion or drug inhibition of Plk4, a kinase essential to centriole biogenesis in both cycling cells and multiciliated cells. Therefore, deuterosomes can be assembled autonomously to mediate de novo centriole amplification in multiciliated cells.
RESUMO
Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signalling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilisation. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely 'non-canonical' substrates. Surprisingly, sequence interrogation of â¼300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both in vitro and in human cells. Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signalling.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/farmacologia , Sulfonas/farmacologia , Linhagem Celular Tumoral , Citometria de Fluxo , Fluorometria , Humanos , Imunoprecipitação , Leupeptinas/farmacologia , Microscopia de Fluorescência , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Espectrometria de Massas em TandemRESUMO
Polo-like kinase 4 (PLK4) is a serine/threonine kinase regulating centriole duplication. CFI-400945 is a highly selective PLK4 inhibitor that deregulates centriole duplication, causing mitotic defects and death of aneuploid cancers. Prior work was substantially extended by showing CFI-400945 causes polyploidy, growth inhibition, and apoptotic death of murine and human lung cancer cells, despite expression of mutated KRAS or p53. Analysis of DNA content by propidium iodide (PI) staining revealed cells with >4N DNA content (polyploidy) markedly increased after CFI-400945 treatment. Centrosome numbers and mitotic spindles were scored. CFI-400945 treatment produced supernumerary centrosomes and mitotic defects in lung cancer cells. In vivo antineoplastic activity of CFI-400945 was established in mice with syngeneic lung cancer xenografts. Lung tumor growth was significantly inhibited at well-tolerated dosages. Phosphohistone H3 staining of resected lung cancers following CFI-400945 treatment confirmed the presence of aberrant mitosis. PLK4 expression profiles in human lung cancers were explored using The Cancer Genome Atlas (TCGA) and RNA in situ hybridization (RNA ISH) of microarrays containing normal and malignant lung tissues. PLK4 expression was significantly higher in the malignant versus normal lung and conferred an unfavorable survival (P < 0.05). Intriguingly, cyclin dependent kinase 2 (CDK2) antagonism cooperated with PLK4 inhibition. Taken together, PLK4 inhibition alone or as part of a combination regimen is a promising way to combat lung cancer.
Assuntos
Apoptose/efeitos dos fármacos , Indazóis/farmacologia , Indóis/farmacologia , Poliploidia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Centrossomo , Regulação Neoplásica da Expressão Gênica , Humanos , Indazóis/uso terapêutico , Indóis/uso terapêutico , Camundongos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismoRESUMO
Glioblastoma (GBM) is one of the most common aggressive cancers of the central nervous system in adults with a high mortality rate. Bortezomib is a boronic acid-based potent proteasome inhibitor that has been actively studied for its anti-tumour effects through inhibition of the proteasome. The proteasome is a key component of the ubiquitin-proteasome pathway that is critical for protein homeostasis, regulation of cellular growth, and apoptosis. Overexpression of polo-like kinase 4 (PLK4) is commonly reported in tumour cells and increases their invasive and metastatic abilities. In this study, we established a cell model of PLK4 knockdown and overexpression in LN-18, A172 and LN-229 cells and found that knockdown of PLK4 expression enhanced the anti-tumour effect of bortezomib. We further found that this effect may be mediated by the PTEN/PI3K/AKT/mTOR signalling pathway and that the apoptotic and oxidative stress processes were activated, while the expression of matrix metalloproteinases (MMPs) was down-regulated. Similar phenomenon was observed using in vitro experiments. Thus, we speculate that PLK4 inhibition may be a new therapeutic strategy for GBM.
Assuntos
Bortezomib/farmacologia , Proliferação de Células/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Proteínas Serina-Treonina Quinases/genética , Animais , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Xenoenxertos , Humanos , Camundongos , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Inibidores de Proteassoma/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genéticaRESUMO
The centrosome, consisting of two centrioles surrounded by a dense network of proteins, is the microtubule-organizing center of animal cells. Polo-like kinase 4 (PLK4) is a Ser/Thr protein kinase and the master regulator of centriole duplication, but it may play additional roles in centrosome function. To identify additional proteins regulated by PLK4, we generated an RPE-1 human cell line with a genetically engineered "analog-sensitive" PLK4AS, which genetically encodes chemical sensitivity to competitive inhibition via a bulky ATP analog. We used this transgenic line in an unbiased multiplex phosphoproteomic screen. Several hits were identified and validated as direct PLK4 substrates by in vitro kinase assays. Among them, we confirmed Ser-78 in centrosomal protein 131 (CEP131, also known as AZI1) as a direct substrate of PLK4. Using immunofluorescence microscopy, we observed that although PLK4-mediated phosphorylation of Ser-78 is dispensable for CEP131 localization, ciliogenesis, and centriole duplication, it is essential for maintaining the integrity of centriolar satellites. We also found that PLK4 inhibition or use of a nonphosphorylatable CEP131 variant results in dispersed centriolar satellites. Moreover, replacement of endogenous WT CEP131 with an S78D phosphomimetic variant promoted aggregation of centriolar satellites. We conclude that PLK4 phosphorylates CEP131 at Ser-78 to maintain centriolar satellite integrity.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Proteínas dos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ciclo Celular/genética , Centríolos/genética , Proteínas do Citoesqueleto , Células HeLa , Humanos , Proteínas dos Microtúbulos/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genéticaRESUMO
The centrosome is an important microtubule-organising centre (MTOC) in animal cells. It consists of two barrel-shaped structures, the centrioles, surrounded by the pericentriolar material (PCM), which nucleates microtubules. Centrosomes can form close to an existing structure (canonical duplication) or de novo How centrosomes form de novo is not known. The master driver of centrosome biogenesis, PLK4, is critical for the recruitment of several centriole components. Here, we investigate the beginning of centrosome biogenesis, taking advantage of Xenopus egg extracts, where PLK4 can induce de novo MTOC formation ( Eckerdt et al., 2011; Zitouni et al., 2016). Surprisingly, we observe that in vitro, PLK4 can self-assemble into condensates that recruit α- and ß-tubulins. In Xenopus extracts, PLK4 assemblies additionally recruit STIL, a substrate of PLK4, and the microtubule nucleator γ-tubulin, forming acentriolar MTOCs de novo The assembly of these robust microtubule asters is independent of dynein, similar to what is found for centrosomes. We suggest a new mechanism of action for PLK4, where it forms a self-organising catalytic scaffold that recruits centriole components, PCM factors and α- and ß-tubulins, leading to MTOC formation.This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Centríolos/metabolismo , Centrossomo/metabolismo , Dineínas/metabolismo , Fuso Acromático/metabolismo , Xenopus laevis/metabolismoRESUMO
Emerging evidence has demonstrated the crucial roles of long noncoding RNAs in human cancers, including neuroblastoma (NB). DLX6 antisense RNA 1 (DLX6-AS1) has been identified as an oncogenic driver in NB. However, the mechanisms of DLX6-AS1 in NB progression are not fully understood. Our data showed that DLX6-AS1 was significantly overexpressed in NB tissues and cells. Moreover, DLX6-AS1 silencing repressed NB cell viability, colony formation, migration, and invasion, and promoted cell cycle arrest and apoptosis in vitro, as well as decreased tumor growth in vivo. Mechanistically, DLX6-AS1 operated as a miR-513c-5p sponge. MiR-513c-5p mediated the regulation of DLX6-AS1 on NB cell malignant progression in vitro. PLK4 was a target of miR-513c-5p- and DLX6-AS1-controlled PLK4 expression via sponging miR-513c-5p. Furthermore, the suppressive effect of miR-513c-5p overexpression on NB cell malignant progression in vitro was reversed by PLK4 upregulation. Our findings identified a novel regulatory mechanism, the DLX6-AS1/miR-513c-5p/PLK4 axis, in NB progression, highlighting a strong rationale for developing DLX6-AS1 as a new target for NB management.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , MicroRNAs/genética , Neuroblastoma/patologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA Antissenso/genética , RNA Longo não Codificante/genética , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neuroblastoma/genética , Neuroblastoma/metabolismo , Prognóstico , Proteínas Serina-Treonina Quinases/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Long noncoding RNA small nucleolar RNA host gene 16 (lncRNA SNHG16) has been revealed to be involved in the tumorigenesis of neuroblastoma. However, the role of SNHG16 in regulating cisplatin sensitivity in neuroblastoma remains largely unknown. METHODS: The expression of SNHG16, microRNA (miR)-338-3p and polo-like kinase 4 (PLK4) mRNA was measured using quantitative real-time polymerase chain reaction. The protein levels of PLK4, multidrug resistance protein 1 (MRP1), multidrug-resistance gene 1-type p-glycoprotein (P-gp) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway-related proteins were detected by Western blot. The half maximal inhibitory concentration (IC50) value, cell proliferation, migration and invasion were analyzed using Cell Counting Kit-8 assays or Transwell assay. Apoptotic cells were measured by Flow cytometry. The interaction between miR-338-3p and SNHG16 or PLK4 was confirmed by dual-luciferase reporter and RNA immunoprecipitation assay. In vivo experiments were conducted through the murine xenograft model. RESULTS: SNHG16 was up-regulated, while miR-338-3p was down-regulated in cisplatin-resistant neuroblastoma tissues and cells. SNHG16 silencing weakened cisplatin resistance, reflected by the reduction of IC50 value, down-regulation of MRP-1 and P-gp protein expression, suppression of proliferation, migration and invasion, as well as enhancement of apoptosis in SNHG16 deletion cisplatin-resistant neuroblastoma cells. Besides that, SNHG16 could regulate PLK4 expression by sponging miR-338-3p and SNHG16/miR-338-3p/PLK4 axis could affect the activation of PI3K/AKT pathway in cisplatin-resistant neuroblastoma cells. MiR-338-3p inhibition attenuated SNHG16 deletion-mediated impairment on cisplatin resistance and PLK4 overexpression reversed the decrease of cisplatin-resistance induced by miR-338-3p re-expression. Furthermore, SNHG16 knockdown contributed to the anti-tumor effect of cisplatin in neuroblastoma in vivo. CONCLUSION: SNHG16 contributed to the tumorigenesis and cisplatin resistance in neuroblastoma possibly through miR-338-3p/PLK4 pathway, indicating a novel insight for overcoming chemoresistance in neuroblastoma patients.
RESUMO
Recent evidence indicates that activation of adenosine monophosphate-activated protein kinase (AMPK), a highly conserved sensor and modulator of cellular energy and redox, regulates cell mitosis. However, the underlying molecular mechanisms for AMPKα subunit regulation of chromosome segregation remain poorly understood. This study aimed to ascertain if AMPKα1 deletion contributes to chromosome missegregation by elevating Polo-like kinase 4 (PLK4) expression. Centrosome proteins and aneuploidy were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J) or AMPKα1 homozygous deficient (AMPKα1-/-) mice by Western blotting and metaphase chromosome spread. Deletion of AMPKα1, the predominant AMPKα isoform in immortalized MEFs, led to centrosome amplification and chromosome missegregation, as well as the consequent aneuploidy (34-66%) and micronucleus. Furthermore, AMPKα1 null cells exhibited a significant induction of PLK4. Knockdown of nuclear factor kappa B2/p52 ameliorated the PLK4 elevation in AMPKα1-deleted MEFs. Finally, PLK4 inhibition by Centrinone reversed centrosome amplification of AMPKα1-deleted MEFs. Taken together, our results suggest that AMPKα1 plays a fundamental role in the maintenance of chromosomal integrity through the control of p52-mediated transcription of PLK4, a trigger of centriole biogenesis.