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1.
Genome Res ; 32(4): 643-655, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35177558

RESUMO

The occurrence and formation of genomic structural variants (SVs) is known to be influenced by the 3D chromatin architecture, but the extent and magnitude have been challenging to study. Here, we apply Hi-C to study chromatin organization before and after induction of chromothripsis in human cells. We use Hi-C to manually assemble the derivative chromosomes following the occurrence of massive complex rearrangements, which allows us to study the sources of SV formation and their consequences on gene regulation. We observe an action-reaction interplay whereby the 3D chromatin architecture directly impacts the location and formation of SVs. In turn, the SVs reshape the chromatin organization to alter the local topologies, replication timing, and gene regulation in cis We show that SVs have a strong tendency to occur between similar chromatin compartments and replication timing regions. Moreover, we find that SVs frequently occur at 3D loop anchors, that SVs can cause a switch in chromatin compartments and replication timing, and that this is a major source of SV-mediated effects on nearby gene expression changes. Finally, we provide evidence for a general mechanistic bias of the 3D chromatin on SV occurrence using data from more than 2700 patient-derived cancer genomes.


Assuntos
Cromotripsia , Genoma , Cromatina/genética , Cromossomos , Genoma Humano , Variação Estrutural do Genoma , Humanos
2.
Nat Commun ; 12(1): 5576, 2021 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-34552071

RESUMO

Chromosome loss that results in monosomy is detrimental to viability, yet it is frequently observed in cancers. How cancers survive with monosomy is unknown. Using p53-deficient monosomic cell lines, we find that chromosome loss impairs proliferation and genomic stability. Transcriptome and proteome analysis demonstrates reduced expression of genes encoded on the monosomes, which is partially compensated in some cases. Monosomy also induces global changes in gene expression. Pathway enrichment analysis reveals that genes involved in ribosome biogenesis and translation are downregulated in all monosomic cells analyzed. Consistently, monosomies display defects in protein synthesis and ribosome assembly. We further show that monosomies are incompatible with p53 expression, likely due to defects in ribosome biogenesis. Accordingly, impaired ribosome biogenesis and p53 inactivation are associated with monosomy in cancer. Our systematic study of monosomy in human cells explains why monosomy is so detrimental and reveals the importance of p53 for monosomy occurrence in cancer.


Assuntos
Monossomia/patologia , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Expressão Gênica , Regulação da Expressão Gênica , Genoma Humano/genética , Instabilidade Genômica , Humanos , Monossomia/genética , Neoplasias/genética , Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
3.
Mol Cancer ; 20(1): 111, 2021 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-34454516

RESUMO

BACKGROUND: Synthetic lethality describes a genetic interaction between two perturbations, leading to cell death, whereas neither event alone has a significant effect on cell viability. This concept can be exploited to specifically target tumor cells. CRISPR viability screens have been widely employed to identify cancer vulnerabilities. However, an approach to systematically infer genetic interactions from viability screens is missing. METHODS: Here we describe PAn-canceR Inferred Synthetic lethalities (PARIS), a machine learning approach to identify cancer vulnerabilities. PARIS predicts synthetic lethal (SL) interactions by combining CRISPR viability screens with genomics and transcriptomics data across hundreds of cancer cell lines profiled within the Cancer Dependency Map. RESULTS: Using PARIS, we predicted 15 high confidence SL interactions within 549 DNA damage repair (DDR) genes. We show experimental validation of an SL interaction between the tumor suppressor CDKN2A, thymidine phosphorylase (TYMP) and the thymidylate synthase (TYMS), which may allow stratifying patients for treatment with TYMS inhibitors. Using genome-wide mapping of SL interactions for DDR genes, we unraveled a dependency between the aldehyde dehydrogenase ALDH2 and the BRCA-interacting protein BRIP1. Our results suggest BRIP1 as a potential therapeutic target in ~ 30% of all tumors, which express low levels of ALDH2. CONCLUSIONS: PARIS is an unbiased, scalable and easy to adapt platform to identify SL interactions that should aid in improving cancer therapy with increased availability of cancer genomics data.


Assuntos
Biologia Computacional/métodos , Aprendizado de Máquina , Modelos Biológicos , Neoplasias/etiologia , Mutações Sintéticas Letais , Linhagem Celular Tumoral , Suscetibilidade a Doenças , Perfilação da Expressão Gênica/métodos , Predisposição Genética para Doença , Genômica/métodos , Humanos , Neoplasias/metabolismo
4.
Cell Rep ; 31(1): 107465, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32268084

RESUMO

TP53 deficiency is the most common alteration in cancer; however, this alone is typically insufficient to drive tumorigenesis. To identify genes promoting tumorigenesis in combination with TP53 deficiency, we perform genome-wide CRISPR-Cas9 knockout screens coupled with proliferation and transformation assays in isogenic cell lines. Loss of several known tumor suppressors enhances cellular proliferation and transformation. Loss of neddylation pathway genes promotes uncontrolled proliferation exclusively in TP53-deficient cells. Combined loss of CUL3 and TP53 activates an oncogenic transcriptional program governed by the nuclear factor κB (NF-κB), AP-1, and transforming growth factor ß (TGF-ß) pathways. This program maintains persistent cellular proliferation, induces partial epithelial to mesenchymal transition, and increases DNA damage, genomic instability, and chromosomal rearrangements. Our findings reveal CUL3 loss as a key event stimulating persistent proliferation in TP53-deficient cells. These findings may be clinically relevant, since TP53-CUL3-deficient cells are highly sensitive to ataxia telangiectasia mutated (ATM) inhibition, exposing a vulnerability that could be exploited for cancer treatment.


Assuntos
Proteínas Culina/genética , Proteína Supressora de Tumor p53/genética , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Carcinogênese/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Proteínas Culina/metabolismo , Transição Epitelial-Mesenquimal , Estudo de Associação Genômica Ampla , Instabilidade Genômica , Humanos , NF-kappa B/metabolismo , Epitélio Pigmentado da Retina/citologia , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismo
5.
Oncotarget ; 11(48): 4490-4503, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-33400734

RESUMO

Tumor cells typically enhance their metabolic capacity to sustain their higher rate of growth and proliferation. One way to elevate the nutrient intake into cancer cells is to increase the expression of genes encoding amino acid transporters, which may represent targetable vulnerabilities. Here, we study the regulation and function of the broad amino acid transporter SLC6A14 in combination with metabolic stress, providing insights into an uncharacterized aspect of the transporter activity. We analyze the pattern of transcriptional changes in a panel of breast cancer cell lines upon metabolic stress and found that SLC6A14 expression levels are increased in the absence of methionine. Methionine deprivation, which can be achieved via modulation of dietary methionine intake in tumor cells, in turn leads to a heightened activation of the AMP-activated kinase (AMPK) in SLC6A14-deficient cells. While SLC6A14 genetic deficiency does not have a major impact on cell proliferation, combined depletion of AMPK and SLC6A14 leads to an increase in apoptosis upon methionine starvation, suggesting that combined targeting of SLC6A14 and AMPK can be exploited as a therapeutic approach to starve tumor cells.

6.
Nat Biotechnol ; 38(3): 343-354, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31873213

RESUMO

Structural variation (SV), involving deletions, duplications, inversions and translocations of DNA segments, is a major source of genetic variability in somatic cells and can dysregulate cancer-related pathways. However, discovering somatic SVs in single cells has been challenging, with copy-number-neutral and complex variants typically escaping detection. Here we describe single-cell tri-channel processing (scTRIP), a computational framework that integrates read depth, template strand and haplotype phase to comprehensively discover SVs in individual cells. We surveyed SV landscapes of 565 single cells, including transformed epithelial cells and patient-derived leukemic samples, to discover abundant SV classes, including inversions, translocations and complex DNA rearrangements. Analysis of the leukemic samples revealed four times more somatic SVs than cytogenetic karyotyping, submicroscopic copy-number alterations, oncogenic copy-neutral rearrangements and a subclonal chromothripsis event. Advancing current methods, single-cell tri-channel processing can directly measure SV mutational processes in individual cells, such as breakage-fusion-bridge cycles, facilitating studies of clonal evolution, genetic mosaicism and SV formation mechanisms, which could improve disease classification for precision medicine.


Assuntos
Biologia Computacional/métodos , Variação Estrutural do Genoma , Leucemia/genética , Análise de Célula Única/métodos , Linhagem Celular , Cromotripsia , Evolução Clonal , Rearranjo Gênico , Humanos , Mutação INDEL , Inversão de Sequência , Translocação Genética
7.
Mol Syst Biol ; 15(12): e8983, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31885201

RESUMO

Arrayed CRISPR-based screens emerge as a powerful alternative to pooled screens making it possible to investigate a wide range of cellular phenotypes that are typically not amenable to pooled screens. Here, we describe a solid-phase transfection platform that enables CRISPR-based genetic screens in arrayed format with flexible readouts. We demonstrate efficient gene knockout upon delivery of guide RNAs and Cas9/guide RNA ribonucleoprotein complexes into untransformed and cancer cell lines. In addition, we provide evidence that our platform can be easily adapted to high-throughput screens and we use this approach to study oncogene addiction in tumor cells. Finally demonstrating that the human primary cells can also be edited using this method, we pave the way for rapid testing of potential targeted therapies.


Assuntos
Edição de Genes/instrumentação , Neoplasias/genética , RNA Guia de Cinetoplastídeos/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Predisposição Genética para Doença , Ensaios de Triagem em Larga Escala , Humanos , Fenótipo , Transfecção
8.
Lancet Oncol ; 19(6): 785-798, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29753700

RESUMO

BACKGROUND: Medulloblastoma is associated with rare hereditary cancer predisposition syndromes; however, consensus medulloblastoma predisposition genes have not been defined and screening guidelines for genetic counselling and testing for paediatric patients are not available. We aimed to assess and define these genes to provide evidence for future screening guidelines. METHODS: In this international, multicentre study, we analysed patients with medulloblastoma from retrospective cohorts (International Cancer Genome Consortium [ICGC] PedBrain, Medulloblastoma Advanced Genomics International Consortium [MAGIC], and the CEFALO series) and from prospective cohorts from four clinical studies (SJMB03, SJMB12, SJYC07, and I-HIT-MED). Whole-genome sequences and exome sequences from blood and tumour samples were analysed for rare damaging germline mutations in cancer predisposition genes. DNA methylation profiling was done to determine consensus molecular subgroups: WNT (MBWNT), SHH (MBSHH), group 3 (MBGroup3), and group 4 (MBGroup4). Medulloblastoma predisposition genes were predicted on the basis of rare variant burden tests against controls without a cancer diagnosis from the Exome Aggregation Consortium (ExAC). Previously defined somatic mutational signatures were used to further classify medulloblastoma genomes into two groups, a clock-like group (signatures 1 and 5) and a homologous recombination repair deficiency-like group (signatures 3 and 8), and chromothripsis was investigated using previously established criteria. Progression-free survival and overall survival were modelled for patients with a genetic predisposition to medulloblastoma. FINDINGS: We included a total of 1022 patients with medulloblastoma from the retrospective cohorts (n=673) and the four prospective studies (n=349), from whom blood samples (n=1022) and tumour samples (n=800) were analysed for germline mutations in 110 cancer predisposition genes. In our rare variant burden analysis, we compared these against 53 105 sequenced controls from ExAC and identified APC, BRCA2, PALB2, PTCH1, SUFU, and TP53 as consensus medulloblastoma predisposition genes according to our rare variant burden analysis and estimated that germline mutations accounted for 6% of medulloblastoma diagnoses in the retrospective cohort. The prevalence of genetic predispositions differed between molecular subgroups in the retrospective cohort and was highest for patients in the MBSHH subgroup (20% in the retrospective cohort). These estimates were replicated in the prospective clinical cohort (germline mutations accounted for 5% of medulloblastoma diagnoses, with the highest prevalence [14%] in the MBSHH subgroup). Patients with germline APC mutations developed MBWNT and accounted for most (five [71%] of seven) cases of MBWNT that had no somatic CTNNB1 exon 3 mutations. Patients with germline mutations in SUFU and PTCH1 mostly developed infant MBSHH. Germline TP53 mutations presented only in childhood patients in the MBSHH subgroup and explained more than half (eight [57%] of 14) of all chromothripsis events in this subgroup. Germline mutations in PALB2 and BRCA2 were observed across the MBSHH, MBGroup3, and MBGroup4 molecular subgroups and were associated with mutational signatures typical of homologous recombination repair deficiency. In patients with a genetic predisposition to medulloblastoma, 5-year progression-free survival was 52% (95% CI 40-69) and 5-year overall survival was 65% (95% CI 52-81); these survival estimates differed significantly across patients with germline mutations in different medulloblastoma predisposition genes. INTERPRETATION: Genetic counselling and testing should be used as a standard-of-care procedure in patients with MBWNT and MBSHH because these patients have the highest prevalence of damaging germline mutations in known cancer predisposition genes. We propose criteria for routine genetic screening for patients with medulloblastoma based on clinical and molecular tumour characteristics. FUNDING: German Cancer Aid; German Federal Ministry of Education and Research; German Childhood Cancer Foundation (Deutsche Kinderkrebsstiftung); European Research Council; National Institutes of Health; Canadian Institutes for Health Research; German Cancer Research Center; St Jude Comprehensive Cancer Center; American Lebanese Syrian Associated Charities; Swiss National Science Foundation; European Molecular Biology Organization; Cancer Research UK; Hertie Foundation; Alexander and Margaret Stewart Trust; V Foundation for Cancer Research; Sontag Foundation; Musicians Against Childhood Cancer; BC Cancer Foundation; Swedish Council for Health, Working Life and Welfare; Swedish Research Council; Swedish Cancer Society; the Swedish Radiation Protection Authority; Danish Strategic Research Council; Swiss Federal Office of Public Health; Swiss Research Foundation on Mobile Communication; Masaryk University; Ministry of Health of the Czech Republic; Research Council of Norway; Genome Canada; Genome BC; Terry Fox Research Institute; Ontario Institute for Cancer Research; Pediatric Oncology Group of Ontario; The Family of Kathleen Lorette and the Clark H Smith Brain Tumour Centre; Montreal Children's Hospital Foundation; The Hospital for Sick Children: Sonia and Arthur Labatt Brain Tumour Research Centre, Chief of Research Fund, Cancer Genetics Program, Garron Family Cancer Centre, MDT's Garron Family Endowment; BC Childhood Cancer Parents Association; Cure Search Foundation; Pediatric Brain Tumor Foundation; Brainchild; and the Government of Ontario.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Cerebelares/genética , Metilação de DNA , Testes Genéticos/métodos , Mutação em Linhagem Germinativa , Meduloblastoma/genética , Modelos Genéticos , Adolescente , Adulto , Neoplasias Cerebelares/mortalidade , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/terapia , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Hereditariedade , Humanos , Lactente , Masculino , Meduloblastoma/mortalidade , Meduloblastoma/patologia , Meduloblastoma/terapia , Linhagem , Fenótipo , Valor Preditivo dos Testes , Intervalo Livre de Progressão , Estudos Prospectivos , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fatores de Risco , Transcriptoma , Sequenciamento do Exoma , Adulto Jovem
9.
Genome Res ; 27(4): 501-511, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28320919

RESUMO

Patterns of gene expression in tumors can arise as a consequence of or result in genomic instability, characterized by the accumulation of somatic copy number alterations (SCNAs) and point mutations (PMs). Expression signatures have been widely used as markers for genomic instability, and both SCNAs and PMs could be thought to associate with distinct signatures given their different formation mechanisms. Here we test this notion by systematically investigating SCNA, PM, and transcriptome data from 2660 cancer patients representing 11 tumor types. Notably, our data indicate that similar expression signatures can be derived from correlating gene expression with either SCNA or PM load. Gene sets related to cell growth and proliferation generally associated positively, and immunoregulatory gene sets negatively, with variant burden. In-depth analyses revealed several genes whose de-regulation correlates with SCNA but not with PM burden, yielding downstream effectors of TP53 and MYC signaling unique to high-SCNA tumors. We compared our findings to expression changes observed in two different cancer mouse models with persistent mitotic chromosomal instability, observing a decrease in proliferative expression signatures. Our results suggest that overexpression of cell-cycle-related genes are a characteristic of proliferation, and likely tumor evolution, rather than ongoing genomic instability.


Assuntos
Aneuploidia , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Transcriptoma , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Variações do Número de Cópias de DNA , Instabilidade Genômica , Humanos , Acúmulo de Mutações , Mutação Puntual
10.
Nat Genet ; 49(1): 65-74, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27869826

RESUMO

Extensive prior research focused on somatic copy-number alterations (SCNAs) affecting cancer genes, yet the extent to which recurrent SCNAs exert their influence through rearrangement of cis-regulatory elements (CREs) remains unclear. Here we present a framework for inferring cancer-related gene overexpression resulting from CRE reorganization (e.g., enhancer hijacking) by integrating SCNAs, gene expression data and information on topologically associating domains (TADs). Analysis of 7,416 cancer genomes uncovered several pan-cancer candidate genes, including IRS4, SMARCA1 and TERT. We demonstrate that IRS4 overexpression in lung cancer is associated with recurrent deletions in cis, and we present evidence supporting a tumor-promoting role. We additionally pursued cancer-type-specific analyses and uncovered IGF2 as a target for enhancer hijacking in colorectal cancer. Recurrent tandem duplications intersecting with a TAD boundary mediate de novo formation of a 3D contact domain comprising IGF2 and a lineage-specific super-enhancer, resulting in high-level gene activation. Our framework enables systematic inference of CRE rearrangements mediating dysregulation in cancer.


Assuntos
Variações do Número de Cópias de DNA/genética , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Substratos do Receptor de Insulina/genética , Fator de Crescimento Insulin-Like II/genética , Neoplasias/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Regiões Promotoras Genéticas
11.
Cell Rep ; 15(12): 2679-91, 2016 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-27292643

RESUMO

Chromosome instability (CIN) is associated with poor survival and therapeutic outcome in a number of malignancies. Despite this correlation, CIN can also lead to growth disadvantages. Here, we show that simultaneous overexpression of the mitotic checkpoint protein Mad2 with Kras(G12D) or Her2 in mammary glands of adult mice results in mitotic checkpoint overactivation and a delay in tumor onset. Time-lapse imaging of organotypic cultures and pathologic analysis prior to tumor establishment reveals error-prone mitosis, mitotic arrest, and cell death. Nonetheless, Mad2 expression persists and increases karyotype complexity in Kras tumors. Faced with the selective pressure of oncogene withdrawal, Mad2-positive tumors have a higher frequency of developing persistent subclones that avoid remission and continue to grow.


Assuntos
Instabilidade Cromossômica , Proteínas Mad2/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Oncogenes , Aneuploidia , Animais , Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Células Cultivadas , Segregação de Cromossomos/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Camundongos , Mitose , Fenótipo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptor ErbB-2 , Fuso Acromático/metabolismo , Imagem com Lapso de Tempo , Transgenes
12.
C R Biol ; 339(7-8): 308-13, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27342254

RESUMO

Characterizing genomic structural variations (SVs) in the human genome remains challenging, and there is a growing interest to understand somatic SVs occurring in cancer, a disease of the genome. A havoc-causing SV process known as chromothripsis scars the genome when localized chromosome shattering and repair occur in a one-off catastrophe. Recent efforts led to the development of a set of conceptual criteria for the inference of chromothripsis events in cancer genomes and to the development of experimental model systems for studying this striking DNA alteration process in vitro. We discuss these approaches, and additionally touch upon current "Big Data" efforts that employ hybrid cloud computing to enable studies of numerous cancer genomes in an effort to search for commonalities and differences in molecular DNA alteration processes in cancer.


Assuntos
Variação Estrutural do Genoma/genética , Neoplasias/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Genoma Humano , Variação Estrutural do Genoma/efeitos dos fármacos , Humanos , Biologia Molecular , Neoplasias/tratamento farmacológico
13.
Genes Chromosomes Cancer ; 55(9): 677-87, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27121553

RESUMO

Congenital gliobastoma multiforme (GBM) is rare and little is known about the molecular defects underlying the initiation and progression of this tumor type. We present a case of congenital GBM analyzed by conventional cytogenetics, fluorescence in situ hybridization, array comparative genomic hybridization and next generation sequencing. On cytogenetic analysis we detected a reciprocal translocation t(6;12)(q21;q24.3). By sequencing, the translocation was shown to form a fusion between the 5' region of ZCCHC8 and the 3' region of ROS1. RT-PCR analyses confirmed the existence of an in-frame fusion transcript with ZCCHC8 exons 1-3 joined to ROS1 exons 36-43. In addition to the ZCCHC8-ROS1 fusion, we detected a deletion in the short arm of chromosome 9, including homozygous loss of the CDKN2A/2B locus in 9p21.3 and heterozygous deletion of the HAUS6 gene in 9p22.1. The latter encodes a protein involved in faithful chromosome segregation by regulating microtubule nucleation and its deletion might be associated with the marked subclonal changes of ploidy observed in the tumor. This report adds the ZCCHC8-ROS1 fusion as oncogenic driver in GBM and supports the role of ROS1 activation in the pathogenesis of a subset of GBM. © 2016 Wiley Periodicals, Inc.


Assuntos
Proteínas de Transporte/genética , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 6/genética , Glioblastoma/congênito , Glioblastoma/genética , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Translocação Genética/genética , Hibridização Genômica Comparativa , Análise Citogenética , Glioblastoma/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Oncotarget ; 7(25): 37608-37621, 2016 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-26993771

RESUMO

High-risk human papillomavirus (hrHPV) types induce immortalization of primary human epithelial cells. Previously we demonstrated that immortalization of human foreskin keratinocytes (HFKs) is HPV type dependent, as reflected by the presence or absence of a crisis period before reaching immortality. This study determined how the immortalization capacity of ten hrHPV types relates to DNA damage induction and overall genomic instability in HFKs.Twenty five cell cultures obtained by transduction of ten hrHPV types (i.e. HPV16/18/31/33/35/45/51/59/66/70 E6E7) in two or three HFK donors each were studied.All hrHPV-transduced HFKs showed an increased number of double strand DNA breaks compared to controls, without exhibiting significant differences between types. However, immortal descendants of HPV-transduced HFKs that underwent a prior crisis period (HPV45/51/59/66/70-transduced HFKs) showed significantly more chromosomal aberrations compared to those without crisis (HPV16/18/31/33/35-transduced HFKs). Notably, the hTERT locus at 5p was exclusively gained in cells with a history of crisis and coincided with increased expression. Chromothripsis was detected in one cell line in which multiple rearrangements within chromosome 8 resulted in a gain of MYC.Together we demonstrated that upon HPV-induced immortalization, the number of chromosomal aberrations is inversely related to the viral immortalization capacity. We propose that hrHPV types with reduced immortalization capacity in vitro, reflected by a crisis period, require more genetic host cell aberrations to facilitate immortalization than types that can immortalize without crisis. This may in part explain the observed differences in HPV-type prevalence in cervical cancers and emphasizes that changes in the host cell genome contribute to HPV-induced carcinogenesis.


Assuntos
Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Infecções por Papillomavirus/virologia , Instabilidade Cromossômica , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 18/metabolismo , Humanos
15.
Mol Syst Biol ; 11(9): 828, 2015 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-26415501

RESUMO

A remarkable observation emerging from recent cancer genome analyses is the identification of chromothripsis as a one-off genomic catastrophe, resulting in massive somatic DNA structural rearrangements (SRs). Largely due to lack of suitable model systems, the mechanistic basis of chromothripsis has remained elusive. We developed an integrative method termed "complex alterations after selection and transformation (CAST)," enabling efficient in vitro generation of complex DNA rearrangements including chromothripsis, using cell perturbations coupled with a strong selection barrier followed by massively parallel sequencing. We employed this methodology to characterize catastrophic SR formation processes, their temporal sequence, and their impact on gene expression and cell division. Our in vitro system uncovered a propensity of chromothripsis to occur in cells with damaged telomeres, and in particular in hyperploid cells. Analysis of primary medulloblastoma cancer genomes verified the link between hyperploidy and chromothripsis in vivo. CAST provides the foundation for mechanistic dissection of complex DNA rearrangement processes.


Assuntos
Cromossomos Humanos/genética , Rearranjo Gênico , Genoma Humano/genética , Instabilidade Genômica/genética , Neoplasias/genética , Aneuploidia , Divisão Celular , Linhagem Celular , Aberrações Cromossômicas , Variações do Número de Cópias de DNA/genética , Humanos , Meduloblastoma/genética , Poliploidia , Telômero/genética , Telômero/patologia , Proteína 2 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo
16.
Philos Trans R Soc Lond B Biol Sci ; 369(1650)2014 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-25047615

RESUMO

The centrosome is the main microtubule (MT)-organizing centre of animal cells. It consists of two centrioles and a multi-layered proteinaceous structure that surrounds the centrioles, the so-called pericentriolar material. Centrosomes promote de novo assembly of MTs and thus play important roles in Golgi organization, cell polarity, cell motility and the organization of the mitotic spindle. To execute these functions, centrosomes have to adopt particular cellular positions. Actin and MT networks and the association of the centrosomes to the nuclear envelope define the correct positioning of the centrosomes. Another important feature of centrosomes is the centrosomal linker that connects the two centrosomes. The centrosome linker assembles in late mitosis/G1 simultaneously with centriole disengagement and is dissolved before or at the beginning of mitosis. Linker dissolution is important for mitotic spindle formation, and its cell cycle timing has profound influences on the execution of mitosis and proficiency of chromosome segregation. In this review, we will focus on the mechanisms of centrosome positioning and separation, and describe their functions and mechanisms in the light of recent findings.


Assuntos
Ciclo Celular/fisiologia , Centríolos/fisiologia , Centrossomo/fisiologia , Microtúbulos/fisiologia , Proteínas Motores Moleculares/metabolismo , Fuso Acromático/fisiologia , Dineínas/metabolismo , Humanos , Cinesinas/metabolismo , Quinases Relacionadas a NIMA , Proteínas Serina-Treonina Quinases/metabolismo
17.
Dev Cell ; 25(3): 229-40, 2013 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-23643362

RESUMO

Timely and accurate assembly of the mitotic spindle is critical for the faithful segregation of chromosomes, and centrosome separation is a key step in this process. The timing of centrosome separation varies dramatically between cell types; however, the mechanisms responsible for these differences and its significance are unclear. Here, we show that activation of epidermal growth factor receptor (EGFR) signaling determines the timing of centrosome separation. Premature separation of centrosomes decreases the requirement for the major mitotic kinesin Eg5 for spindle assembly, accelerates mitosis, and decreases the rate of chromosome missegregation. Importantly, EGF stimulation impacts upon centrosome separation and mitotic progression to different degrees in different cell lines. Cells with high EGFR levels fail to arrest in mitosis upon Eg5 inhibition. This has important implications for cancer therapy because cells with high centrosomal response to EGF are more susceptible to combinatorial inhibition of EGFR and Eg5.


Assuntos
Centrossomo/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Mitose/efeitos dos fármacos , Proliferação de Células , Sobrevivência Celular , Cisteína/análogos & derivados , Cisteína/farmacologia , Replicação do DNA , Ativação Enzimática , Células HeLa , Humanos , Cinesinas/antagonistas & inibidores , Cinesinas/genética , Cinesinas/metabolismo , Quinases Relacionadas a NIMA , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular , Serina-Treonina Quinase 3 , Transdução de Sinais , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/genética , Fuso Acromático/metabolismo , Fatores de Tempo , Transfecção
18.
J Cell Biol ; 197(1): 11-8, 2012 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-22472437

RESUMO

The centrosome, which consists of two centrioles and the surrounding pericentriolar material, is the primary microtubule-organizing center (MTOC) in animal cells. Like chromosomes, centrosomes duplicate once per cell cycle and defects that lead to abnormalities in the number of centrosomes result in genomic instability, a hallmark of most cancer cells. Increasing evidence suggests that the separation of the two centrioles (disengagement) is required for centrosome duplication. After centriole disengagement, a proteinaceous linker is established that still connects the two centrioles. In G2, this linker is resolved (centrosome separation), thereby allowing the centrosomes to separate and form the poles of the bipolar spindle. Recent work has identified new players that regulate these two processes and revealed unexpected mechanisms controlling the centrosome cycle.


Assuntos
Centrossomo/metabolismo , Animais , Centríolos/metabolismo , Humanos , Cinesinas/metabolismo , Quinases Relacionadas a NIMA , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Xenopus/metabolismo
19.
Curr Biol ; 21(13): 1145-51, 2011 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-21723128

RESUMO

In human cells, separation of the centrosomes and formation of a bipolar spindle are essential for correct chromosome segregation [1]. During interphase, centrosomes are joined together by the linker proteins C-Nap1 and rootletin [2-4]. At the onset of mitosis, these linker proteins are phosphorylated and displaced from centrosomes by the Nek2A kinase, which is regulated by two Hippo pathway components, Mst2 kinase and the scaffold protein hSav1. The kinesin-5 motor protein Eg5 promotes centrosome separation in a parallel pathway to Nek2A [5]. Here, we report that Polo-like kinase 1 (Plk1) functions upstream of the Mst2-Nek2A kinase module in centrosome disjunction as well as being important for Eg5 localization at centrosomes. Plk1 regulates Mst2-Nek2A-induced centrosome disjunction by phosphorylating Mst2. The absence of Plk1 phosphorylation of Mst2 promotes assembly of Nek2A-PP1γ-Mst2 complexes, in which PP1γ counteracts Nek2A kinase activity. In contrast, Plk1 phosphorylation of Mst2 prevents PP1γ binding to Mst2-Nek2A, allowing Nek2A activity to promote centrosome disjunction. We propose that centrosome disjunction is regulated by Plk1, providing a well-balanced control between the counteracting Nek2A and PP1γ activities on the centrosome linker.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Centrossomo/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Células HEK293 , Humanos , Cinesinas/metabolismo , Mitose , Quinases Relacionadas a NIMA , Fosforilação , Proteína Fosfatase 1/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Serina-Treonina Quinase 3 , Quinase 1 Polo-Like
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