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1.
Nat Commun ; 12(1): 4919, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389725

RESUMO

BRCA1 or BRCA2 germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification and BRCA1/2 mutations are mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of ß-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed ß-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression of CDKN1A gene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, ß-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that ß-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Oncogenes/genética , Transcrição Genética/genética , beta Catenina/genética , Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Feminino , Perfilação da Expressão Gênica/métodos , Células HeLa , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , RNA-Seq/métodos , beta Catenina/metabolismo
2.
Nucleic Acids Res ; 46(10): 4903-4918, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29718321

RESUMO

The replicative immortality of human cancer cells is achieved by activation of a telomere maintenance mechanism (TMM). To achieve this, cancer cells utilise either the enzyme telomerase, or the Alternative Lengthening of Telomeres (ALT) pathway. These distinct molecular pathways are incompletely understood with respect to activation and propagation, as well as their associations with clinical outcomes. We have identified significant differences in the telomere repeat composition of tumours that use ALT compared to tumours that do not. We then employed a machine learning approach to stratify tumours according to telomere repeat content with an accuracy of 91.6%. Importantly, this classification approach is applicable across all tumour types. Analysis of pathway mutations that were under-represented in ALT tumours, across 1,075 tumour samples, revealed that the autophagy, cell cycle control of chromosomal replication, and transcriptional regulatory network in embryonic stem cells pathways are involved in the survival of ALT tumours. Overall, our approach demonstrates that telomere sequence content can be used to stratify ALT activity in cancers, and begin to define the molecular pathways involved in ALT activation.


Assuntos
Biologia Computacional/métodos , Neoplasias/genética , Homeostase do Telômero/genética , Telômero/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Correpressoras , Bases de Dados Genéticas , Feminino , Humanos , Aprendizado de Máquina , Melanoma/genética , Melanoma/mortalidade , Chaperonas Moleculares , Mutação , Neoplasias/mortalidade , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Análise de Sobrevida , Telomerase/genética , Sequenciamento Completo do Exoma , Proteína Nuclear Ligada ao X/genética
3.
Cell Rep ; 19(12): 2544-2556, 2017 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-28636942

RESUMO

Acquisition of replicative immortality is currently regarded as essential for malignant transformation. This is achieved by activating a telomere lengthening mechanism (TLM), either telomerase or alternative lengthening of telomeres, to counter normal telomere attrition. However, a substantial proportion of some cancer types, including glioblastomas, liposarcomas, retinoblastomas, and osteosarcomas, are reportedly TLM-negative. As serial samples of human tumors cannot usually be obtained to monitor telomere length changes, it has previously been impossible to determine whether tumors are truly TLM-deficient, there is a previously unrecognized TLM, or the assay results are false-negative. Here, we show that a subset of high-risk neuroblastomas (with ∼50% 5-year mortality) lacked significant TLM activity. Cancer cells derived from these highly aggressive tumors initially had long telomeres and proliferated for >200 population doublings with ever-shorter telomeres. This indicates that prevention of telomere shortening is not always required for oncogenesis, which has implications for inhibiting TLMs for cancer therapy.


Assuntos
Proliferação de Células , Encurtamento do Telômero , Linhagem Celular Tumoral , Ativação Enzimática , Amplificação de Genes , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Telomerase/metabolismo
4.
Nature ; 545(7653): 175-180, 2017 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-28467829

RESUMO

Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. Here we report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. However, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequences was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all; however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. Most melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals diverse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.


Assuntos
Genoma Humano/genética , Melanoma/genética , Mutação/genética , DNA Helicases/genética , GTP Fosfo-Hidrolases/genética , Genes p16 , Humanos , Melanoma/classificação , Proteínas de Membrana/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Neurofibromatose 1/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Fatores de Processamento de RNA/genética , Transdução de Sinais/efeitos dos fármacos , Telomerase/genética , Telômero/genética , Proteína Supressora de Tumor p53/genética , Raios Ultravioleta/efeitos adversos , Proteína Nuclear Ligada ao X
5.
Methods ; 114: 74-84, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-27595911

RESUMO

The C-Circle Assay has satisfied the need for a rapid, robust and quantitative ALT assay that responds quickly to changes in ALT activity. The C-Circle Assay involves (i) extraction or simple preparation (Quick C-Circle Preparation) of the cell's DNA, which includes C-Circles (ii) amplification of the self-primed C-Circles with a rolling circle amplification reaction and (iii) sequence specific detection of the amplification products by native telomeric DNA dot blot or telomeric qPCR. Here we detail the protocols and considerations required to perform the C-Circle Assay and its controls, which include exonuclease removal of linear telomeric DNA, production of the synthetic C-Circle C96 and modulation of ALT activity by γ-irradiation.


Assuntos
Biomarcadores Tumorais/genética , DNA Circular/análise , DNA de Neoplasias/genética , Neoplasias/diagnóstico , Telômero , Humanos , Neoplasias/genética
6.
J Neurooncol ; 119(1): 17-26, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24792489

RESUMO

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere length maintenance mechanism that enables the unlimited proliferation of a subset of cancer cells. Some neuroblastoma (NB) tumors appear to maintain telomere length by activating ALT. Of 40 NB cell lines, we identified four potential ALT cell lines (CHLA-90, SK-N-FI, LA-N-6, and COG-N-291) that were telomerase-negative and had long telomeres (a feature of ALT cells). All four cell lines lacked MYCN amplification and were p53 non-functional upon irradiation. Two of these cell lines (CHLA-90 and SK-N-FI) were positive for C-circles (telomeric DNA circles) and ALT-associated promyelocytic leukemia nuclear bodies, both of which are phenotypic characteristics of ALT. Mutation of ATRX (associated with ALT in tumors) was only found in CHLA-90. Thus, the ALT phenotype in NB may not be limited to tumors with ATRX mutations but is associated with a lack of MYCN amplification and alterations in the p53 pathway.


Assuntos
Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Transdução de Sinais/genética , Homeostase do Telômero , Telômero/genética , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Amplificação de Genes , Humanos , Proteína Proto-Oncogênica N-Myc , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Telômero/metabolismo , Proteína Supressora de Tumor p53/metabolismo
7.
Nucleic Acids Res ; 42(3): 1733-46, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24225324

RESUMO

Telomeres are terminal repetitive DNA sequences on chromosomes, and are considered to comprise almost exclusively hexameric TTAGGG repeats. We have evaluated telomere sequence content in human cells using whole-genome sequencing followed by telomere read extraction in a panel of mortal cell strains and immortal cell lines. We identified a wide range of telomere variant repeats in human cells, and found evidence that variant repeats are generated by mechanistically distinct processes during telomerase- and ALT-mediated telomere lengthening. Telomerase-mediated telomere extension resulted in biased repeat synthesis of variant repeats that differed from the canonical sequence at positions 1 and 3, but not at positions 2, 4, 5 or 6. This indicates that telomerase is most likely an error-prone reverse transcriptase that misincorporates nucleotides at specific positions on the telomerase RNA template. In contrast, cell lines that use the ALT pathway contained a large range of variant repeats that varied greatly between lines. This is consistent with variant repeats spreading from proximal telomeric regions throughout telomeres in a stochastic manner by recombination-mediated templating of DNA synthesis. The presence of unexpectedly large numbers of variant repeats in cells utilizing either telomere maintenance mechanism suggests a conserved role for variant sequences at human telomeres.


Assuntos
Homeostase do Telômero , Telômero/química , Linhagem Celular , Variação Genética , Humanos , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA , Telomerase/metabolismo
8.
Nucleic Acids Res ; 41(2): e34, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22923525

RESUMO

Alternative lengthening of telomeres (ALT) is one of the two known telomere length maintenance mechanisms that are essential for the unlimited proliferation potential of cancer cells. Existing methods for detecting ALT in tumors require substantial amounts of tumor material and are labor intensive, making it difficult to study prevalence and prognostic significance of ALT in large tumor cohorts. Here, we present a novel strategy utilizing telomere quantitative PCR to diagnose ALT. The protocol is more rapid than conventional methods and scrutinizes two distinct characteristics of ALT cells concurrently: long telomeres and the presence of C-circles (partially double-stranded circles of telomeric C-strand DNA). Requiring only 30 ng of genomic DNA, this protocol will facilitate large-scale studies of ALT in tumors and can be readily adopted by clinical laboratories.


Assuntos
Neoplasias/genética , Reação em Cadeia da Polimerase/métodos , Homeostase do Telômero , Linhagem Celular Tumoral , DNA de Neoplasias/análise , Humanos , Sondas de Oligonucleotídeos , Telômero/química
9.
PLoS One ; 7(11): e50062, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23185534

RESUMO

Alternative Lengthening of Telomeres (ALT) is a non-telomerase mechanism of telomere lengthening that occurs in about 10% of cancers overall and is particularly common in astrocytic brain tumors and specific types of sarcomas. Somatic cell hybridization analyses have previously shown that normal telomerase-negative fibroblasts and telomerase-positive immortalized cell lines contain repressors of ALT activity, indicating that activation of ALT results from loss of one or more unidentified repressors. More recently, ATRX or DAXX was shown to be mutated both in tumors with telomere lengths suggestive of ALT activity and in ALT cell lines. Here, an ALT cell line was separately fused to each of four telomerase-positive cell lines, and four or five independent hybrid lines from each fusion were examined for expression of ATRX and DAXX and for telomere lengthening mechanism. The hybrid lines expressed either telomerase or ALT, with the other mechanism being repressed. DAXX was expressed normally in all parental cell lines and in all of the hybrids. ATRX was expressed normally in each of the four telomerase-positive parental cell lines and in every telomerase-positive hybrid line, and was abnormal in the ALT parental cells and in all but one of the ALT hybrids. This correlation between ALT activity and loss of ATRX expression is consistent with ATRX being a repressor of ALT.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , DNA Helicases/genética , Regulação da Expressão Gênica , Células Híbridas/metabolismo , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Telômero , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fusão Celular , Proteínas Correpressoras , DNA Helicases/metabolismo , Feminino , Humanos , Células Híbridas/patologia , Masculino , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Telomerase/genética , Telomerase/metabolismo , Homeostase do Telômero/genética , Proteína Nuclear Ligada ao X
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