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1.
Gut ; 71(5): 961-973, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-33849943

RESUMO

OBJECTIVE: Recent studies have found aristaless-related homeobox gene (ARX)/pancreatic and duodenal homeobox 1 (PDX1), alpha-thalassemia/mental retardation X-linked (ATRX)/death domain-associated protein (DAXX) and alternative lengthening of telomeres (ALT) to be promising prognostic biomarkers for non-functional pancreatic neuroendocrine tumours (NF-PanNETs). However, they have not been comprehensively evaluated, especially among small NF-PanNETs (≤2.0 cm). Moreover, their status in neuroendocrine tumours (NETs) from other sites remains unknown. DESIGN: An international cohort of 1322 NETs was evaluated by immunolabelling for ARX/PDX1 and ATRX/DAXX, and telomere-specific fluorescence in situ hybridisation for ALT. This cohort included 561 primary NF-PanNETs, 107 NF-PanNET metastases and 654 primary, non-pancreatic non-functional NETs and NET metastases. The results were correlated with numerous clinicopathological features including relapse-free survival (RFS). RESULTS: ATRX/DAXX loss and ALT were associated with several adverse prognostic findings and distant metastasis/recurrence (p<0.001). The 5-year RFS rates for patients with ATRX/DAXX-negative and ALT-positive NF-PanNETs were 40% and 42% as compared with 85% and 86% for wild-type NF-PanNETs (p<0.001 and p<0.001). Shorter 5-year RFS rates for ≤2.0 cm NF-PanNETs patients were also seen with ATRX/DAXX loss (65% vs 92%, p=0.003) and ALT (60% vs 93%, p<0.001). By multivariate analysis, ATRX/DAXX and ALT status were independent prognostic factors for RFS. Conversely, classifying NF-PanNETs by ARX/PDX1 expression did not independently correlate with RFS. Except for 4% of pulmonary carcinoids, ATRX/DAXX loss and ALT were only identified in primary (25% and 29%) and NF-PanNET metastases (62% and 71%). CONCLUSIONS: ATRX/DAXX and ALT should be considered in the prognostic evaluation of NF-PanNETs including ≤2.0 cm tumours, and are highly specific for pancreatic origin among NET metastases of unknown primary.


Assuntos
Deficiência Intelectual , Tumores Neuroendócrinos , Neoplasias Pancreáticas , Talassemia alfa , Proteínas Correpressoras/genética , Genes Homeobox , Proteínas de Homeodomínio , Humanos , Deficiência Intelectual/genética , Chaperonas Moleculares/genética , Recidiva Local de Neoplasia/genética , Tumores Neuroendócrinos/genética , Proteínas Nucleares/genética , Neoplasias Pancreáticas/patologia , Telômero/genética , Telômero/patologia , Fatores de Transcrição/genética , Proteína Nuclear Ligada ao X/genética , Talassemia alfa/genética
2.
Cell Rep ; 37(10): 110088, 2021 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-34879271

RESUMO

Alternative lengthening of telomeres (ALT) is a telomere-elongation mechanism observed in ∼15% of cancer subtypes. Current models indicate that ALT is mediated by homology-directed repair mechanisms. By disrupting MSH6 gene expression, we show that the deficiency of MutSα (MSH2/MSH6) DNA mismatch repair complex causes striking telomere hyperextension. Mechanistically, we show MutSα is specifically recruited to telomeres in ALT cells by associating with the proliferating-cell nuclear antigen (PCNA) subunit of the ALT telomere replisome. We also provide evidence that MutSα counteracts Bloom (BLM) helicase, which adopts a crucial role in stabilizing hyper-extended telomeres and maintaining the survival of MutSα-deficient ALT cancer cells. Lastly, we propose a model in which MutSα deficiency impairs heteroduplex rejection, leading to premature initiation of telomere DNA synthesis that coincides with an accumulation of telomere variant repeats (TVRs). These findings provide evidence that the MutSα DNA mismatch repair complex acts to restrain unwarranted ALT.


Assuntos
DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Neoplasias/enzimologia , Ácidos Nucleicos Heteroduplexes/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Linhagem Celular Tumoral , Reparo de Erro de Pareamento de DNA , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica , Células HeLa , Humanos , Modelos Genéticos , Proteína 2 Homóloga a MutS/genética , Neoplasias/genética , Neoplasias/patologia , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Telômero/genética
3.
Mol Cell ; 81(12): 2640-2655.e8, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34019811

RESUMO

ARH3/ADPRHL2 and PARG are the primary enzymes reversing ADP-ribosylation in vertebrates, yet their functions in vivo remain unclear. ARH3 is the only hydrolase able to remove serine-linked mono(ADP-ribose) (MAR) but is much less efficient than PARG against poly(ADP-ribose) (PAR) chains in vitro. Here, by using ARH3-deficient cells, we demonstrate that endogenous MARylation persists on chromatin throughout the cell cycle, including mitosis, and is surprisingly well tolerated. Conversely, persistent PARylation is highly toxic and has distinct physiological effects, in particular on active transcription histone marks such as H3K9ac and H3K27ac. Furthermore, we reveal a synthetic lethal interaction between ARH3 and PARG and identify loss of ARH3 as a mechanism of PARP inhibitor resistance, both of which can be exploited in cancer therapy. Finally, we extend our findings to neurodegeneration, suggesting that patients with inherited ARH3 deficiency suffer from stress-induced pathogenic increase in PARylation that can be mitigated by PARP inhibition.


Assuntos
Glicosídeo Hidrolases/metabolismo , Poli ADP Ribosilação/fisiologia , ADP-Ribosilação , Adenosina Difosfato Ribose/metabolismo , Linhagem Celular Tumoral , Cromatina , DNA , Dano ao DNA , Fibroblastos/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/fisiologia , Células HEK293 , Células HeLa , Humanos , Poli Adenosina Difosfato Ribose/metabolismo , Cultura Primária de Células
4.
Nat Struct Mol Biol ; 27(12): 1152-1164, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33046907

RESUMO

The synthesis of poly(ADP-ribose) (PAR) reconfigures the local chromatin environment and recruits DNA-repair complexes to damaged chromatin. PAR degradation by poly(ADP-ribose) glycohydrolase (PARG) is essential for progression and completion of DNA repair. Here, we show that inhibition of PARG disrupts homology-directed repair (HDR) mechanisms that underpin alternative lengthening of telomeres (ALT). Proteomic analyses uncover a new role for poly(ADP-ribosyl)ation (PARylation) in regulating the chromatin-assembly factor HIRA in ALT cancer cells. We show that HIRA is enriched at telomeres during the G2 phase and is required for histone H3.3 deposition and telomere DNA synthesis. Depletion of HIRA elicits systemic death of ALT cancer cells that is mitigated by re-expression of ATRX, a protein that is frequently inactivated in ALT tumors. We propose that PARylation enables HIRA to fulfill its essential role in the adaptive response to ATRX deficiency that pervades ALT cancers.


Assuntos
DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Glicosídeo Hidrolases/genética , Poli(ADP-Ribose) Polimerases/genética , Processamento de Proteína Pós-Traducional , Reparo de DNA por Recombinação , Telômero/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Cromatina/ultraestrutura , Dano ao DNA , DNA de Neoplasias/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fase G2 , Glicosídeo Hidrolases/metabolismo , Células HeLa , Chaperonas de Histonas/antagonistas & inibidores , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Poli ADP Ribosilação , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Telômero/ultraestrutura , Homeostase do Telômero , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo
6.
Curr Opin Genet Dev ; 60: 31-40, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32145504

RESUMO

There is unequivocal evidence that telomeres are crucial for cellular homeostasis and that telomere dysfunction can elicit genome instability and potentially initiate events that culminate in cancer. Mounting evidence points to telomeres having a crucial role in driving local and systemic structural rearrangements that drive cancer. These include the classical 'breakage-fusion-bridge' (BFB) cycles and more recently identified genome re-shaping events like kataegis and chromothripsis. In this brief review, we outline the established and most recent advances describing the roles that telomere dysfunction has in the origin of these catastrophic genome rearrangements. We discuss how local and systemic structural rearrangements enable telomere length maintenance, by either telomerase or the alternative lengthening of telomeres, that is essential to sustain cancer cell proliferation.


Assuntos
Transformação Celular Neoplásica/patologia , Genoma Humano , Instabilidade Genômica , Neoplasias/genética , Neoplasias/patologia , Homeostase do Telômero , Telômero , Proliferação de Células , Transformação Celular Neoplásica/genética , Humanos
7.
Trends Cancer ; 6(3): 247-260, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32101727

RESUMO

Alternative lengthening of telomeres (ALT) is a mechanism of telomere maintenance that is observed in many of the most recalcitrant cancer subtypes. Telomeres in ALT cancer cells exhibit a distinctive nucleoprotein architecture shaped by the mismanagement of chromatin that fosters cycles of DNA damage and replicative stress that activate homology-directed repair (HDR). Mutations in specific chromatin-remodeling factors appear to be key determinants of the emergence and survival of ALT cancer cells. However, these may represent vulnerabilities for the targeted elimination of ALT cancer cells that infiltrate tissues and organs to become devastating tumors. In this review we examine recent findings that provide new insights into the factors and mechanisms that mediate telomere length maintenance and survival of ALT cancer cells.


Assuntos
Neoplasias/genética , Homeostase do Telômero , Cromatina/ultraestrutura , Evolução Clonal , Proteínas Correpressoras/antagonistas & inibidores , Proteínas Correpressoras/fisiologia , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA de Neoplasias/metabolismo , DNA de Neoplasias/ultraestrutura , Histonas/fisiologia , Recombinação Homóloga , Humanos , Modelos Genéticos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/fisiologia , Mutação , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Neoplasias/ultraestrutura , Conformação de Ácido Nucleico , Telomerase/genética , Telomerase/fisiologia , Proteína Nuclear Ligada ao X/antagonistas & inibidores , Proteína Nuclear Ligada ao X/fisiologia
9.
Nucleic Acids Res ; 47(19): 10151-10165, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31665741

RESUMO

RAD51 plays a central role in homologous recombination during double-strand break repair and in replication fork dynamics. Misregulation of RAD51 is associated with genetic instability and cancer. RAD51 is regulated by many accessory proteins including the highly conserved Shu complex. Here, we report the function of the human Shu complex during replication to regulate RAD51 recruitment to DNA repair foci and, secondly, during replication fork restart following replication fork stalling. Deletion of the Shu complex members, SWS1 and SWSAP1, using CRISPR/Cas9, renders cells specifically sensitive to the replication fork stalling and collapse caused by methyl methanesulfonate and mitomycin C exposure, a delayed and reduced RAD51 response, and fewer sister chromatid exchanges. Our additional analysis identified SPIDR and PDS5B as novel Shu complex interacting partners and genetically function in the same pathway upon DNA damage. Collectively, our study uncovers a protein complex, which consists of SWS1, SWSAP1, SPIDR and PDS5B, involved in DNA repair and provides insight into Shu complex function and composition.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , Proteínas Nucleares/genética , Recombinases Rec A/genética , Fatores de Transcrição/genética , Sistemas CRISPR-Cas/genética , Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Complexos Multiproteicos/genética , Rad51 Recombinase/genética , Troca de Cromátide Irmã/genética
10.
Mol Cell ; 76(1): 11-26.e7, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31400850

RESUMO

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in aggressive cancers. We show that the disruption of RAD51-associated protein 1 (RAD51AP1) in ALT+ cancer cells leads to generational telomere shortening. This is due to RAD51AP1's involvement in RAD51-dependent homologous recombination (HR) and RAD52-POLD3-dependent break induced DNA synthesis. RAD51AP1 KO ALT+ cells exhibit telomere dysfunction and cytosolic telomeric DNA fragments that are sensed by cGAS. Intriguingly, they activate ULK1-ATG7-dependent autophagy as a survival mechanism to mitigate DNA damage and apoptosis. Importantly, RAD51AP1 protein levels are elevated in ALT+ cells due to MMS21 associated SUMOylation. Mutation of a single SUMO-targeted lysine residue perturbs telomere dynamics. These findings indicate that RAD51AP1 is an essential mediator of the ALT mechanism and is co-opted by post-translational mechanisms to maintain telomere length and ensure proliferation of ALT+ cancer cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Neoplasias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Autofagia , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proliferação de Células , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Recombinação Homóloga , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligases/genética , Ligases/metabolismo , Lisina , Neoplasias/genética , Neoplasias/patologia , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Estabilidade Proteica , Proteínas de Ligação a RNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Transdução de Sinais , Sumoilação , Telômero/genética , Telômero/patologia
11.
Cells ; 8(7)2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31336873

RESUMO

Cancer cells need to acquire telomere maintenance mechanisms in order to counteract progressive telomere shortening due to multiple rounds of replication. Most human tumors maintain their telomeres expressing telomerase whereas the remaining 15%-20% utilize the alternative lengthening of telomeres (ALT) pathway. Previous studies have demonstrated that ionizing radiations (IR) are able to modulate telomere lengths and to transiently induce some of the ALT-pathway hallmarks in normal primary fibroblasts. In the present study, we investigated the telomere length modulation kinetics, telomeric DNA damage induction, and the principal hallmarks of ALT over a period of 13 days in X-ray-exposed primary cells. Our results show that X-ray-treated cells primarily display telomere shortening and telomeric damage caused by persistent IR-induced oxidative stress. After initial telomere erosion, we observed a telomere elongation that was associated to the transient activation of a homologous recombination (HR) based mechanism, sharing several features with the ALT pathway observed in cancer cells. Data indicate that telomeric damage activates telomeric HR-mediated repair in primary cells. The characterization of HR-mediated telomere repair in normal cells may contribute to the understanding of the ALT pathway and to the identification of novel strategies in the treatment of ALT-positive cancers.


Assuntos
Reparo do DNA/fisiologia , DNA/metabolismo , Fibroblastos , Homeostase do Telômero , Encurtamento do Telômero , Telômero/metabolismo , Linhagem Celular , Dano ao DNA , Fibroblastos/citologia , Fibroblastos/metabolismo , Recombinação Homóloga , Humanos , Raios X
12.
Immunity ; 50(1): 51-63.e5, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30635239

RESUMO

Interferon-inducible human oligoadenylate synthetase-like (OASL) and its mouse ortholog, Oasl2, enhance RNA-sensor RIG-I-mediated type I interferon (IFN) induction and inhibit RNA virus replication. Here, we show that OASL and Oasl2 have the opposite effect in the context of DNA virus infection. In Oasl2-/- mice and OASL-deficient human cells, DNA viruses such as vaccinia, herpes simplex, and adenovirus induced increased IFN production, which resulted in reduced virus replication and pathology. Correspondingly, ectopic expression of OASL in human cells inhibited IFN induction through the cGAS-STING DNA-sensing pathway. cGAS was necessary for the reduced DNA virus replication observed in OASL-deficient cells. OASL directly and specifically bound to cGAS independently of double-stranded DNA, resulting in a non-competitive inhibition of the second messenger cyclic GMP-AMP production. Our findings define distinct mechanisms by which OASL differentially regulates host IFN responses during RNA and DNA virus infection and identify OASL as a negative-feedback regulator of cGAS.


Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Infecções por Vírus de DNA/imunologia , Vírus de DNA/fisiologia , Infecções por Vírus de RNA/imunologia , Vírus de RNA/imunologia , 2',5'-Oligoadenilato Sintetase/genética , Animais , AMP Cíclico/metabolismo , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleotidiltransferases/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais , Células THP-1 , Replicação Viral
13.
JCO Precis Oncol ; 3: 1-20, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35100718

RESUMO

PURPOSE: Telomere maintenance is a hallmark of high-risk neuroblastoma; however, the contribution of telomerase and alternative lengthening of telomeres (ALT) to clinical phenotypes has remained unclear. We aimed to determine the clinical relevance of telomerase activation versus ALT as biomarkers in pretreatment neuroblastoma and to assess the potential value of telomerase as a therapeutic target. MATERIALS AND METHODS: The genomic status of TERT and MYCN was assessed in 457 pretreatment neuroblastomas by fluorescence in situ hybridization. ALT was examined in 273 of 457 tumors by detection of ALT-associated promyelocytic leukemia nuclear bodies, and TERT expression was determined by microarrays in 223 of these. Cytotoxic effects of telomerase-interacting compounds were analyzed in neuroblastoma cell lines in vitro and in vivo. RESULTS: We detected TERT rearrangements in 46 of 457 cases (10.1%), MYCN amplification in 93 of 457 cases (20.4%), and elevated TERT expression in tumors lacking TERT or MYCN alterations in 10 of 223 cases (4.5%). ALT activation was found in 49 of 273 cases (17.9%). All these alterations occurred almost mutually exclusively and were associated with unfavorable prognostic variables and adverse outcome. The presence of activated telomerase (ie, TERT rearrangements, MYCN amplification, or high TERT expression without these alterations) was associated with poorest overall survival and was an independent prognostic marker in multivariable analyses. We also found that the telomerase-interacting compound 6-thio-2'-deoxyguanosine effectively inhibited viability and proliferation of neuroblastoma cells bearing activated telomerase. Similarly, tumor growth was strongly impaired upon 6-thio-2'-deoxyguanosine treatment in telomerase-positive neuroblastoma xenografts in mice. CONCLUSION: Our data suggest telomerase activation and ALT define distinct neuroblastoma subgroups with adverse outcome and that telomerase may represent a promising therapeutic target in many high-risk neuroblastomas.

14.
Science ; 362(6419): 1165-1170, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30523111

RESUMO

Neuroblastoma is a pediatric tumor of the sympathetic nervous system. Its clinical course ranges from spontaneous tumor regression to fatal progression. To investigate the molecular features of the divergent tumor subtypes, we performed genome sequencing on 416 pretreatment neuroblastomas and assessed telomere maintenance mechanisms in 208 of these tumors. We found that patients whose tumors lacked telomere maintenance mechanisms had an excellent prognosis, whereas the prognosis of patients whose tumors harbored telomere maintenance mechanisms was substantially worse. Survival rates were lowest for neuroblastoma patients whose tumors harbored telomere maintenance mechanisms in combination with RAS and/or p53 pathway mutations. Spontaneous tumor regression occurred both in the presence and absence of these mutations in patients with telomere maintenance-negative tumors. On the basis of these data, we propose a mechanistic classification of neuroblastoma that may benefit the clinical management of patients.


Assuntos
Neuroblastoma/classificação , Neuroblastoma/mortalidade , Homeostase do Telômero/genética , Criança , Pré-Escolar , Intervalo Livre de Doença , Exoma/genética , Genoma Humano , Humanos , Redes e Vias Metabólicas/genética , Mutação , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Prognóstico , Análise de Sequência de DNA , Proteína Supressora de Tumor p53/genética , Proteínas ras/genética
15.
Gastroenterology ; 154(8): 2060-2063.e8, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29486199

RESUMO

Despite prognostic grading and staging systems, it is a challenge to predict outcomes for patients with pancreatic neuroendocrine tumors (PanNETs). Sequencing studies of PanNETs have identified alterations in death domain-associated protein (DAXX) and alpha-thalassemia/mental retardation X-linked chromatin remodeler (ATRX). In tumors, mutations in DAXX or ATRX and corresponding loss of protein expression correlate with shorter times of disease-free survival and disease-specific survival of patients. However, DAXX or ATRX proteins were lost in only 50% of distant metastases analyzed. We performed whole-exome sequencing analyses of 20 distant metastases from 20 patients with a single nonsyndrome, nonfunctional PanNET. We found distant metastases contained alterations in multiple endocrine neoplasia type 1 (MEN1) (n = 8), ATRX (n = 5), DAXX (n = 5), TSC2 (n = 3), and DEP domain containing 5 (DEPDC5) (n = 3). We found copy number loss of cyclin dependent kinase inhibitor 2A (CDKN2A) in 15 metastases (75%) and alterations in genes that regulate chromatin remodeling, including set domain containing 2 (SETD2) (n = 4), AT-rich interaction domain 1A (ARID1A) (n = 2), chromodomain helicase DNA binding protein 8 (CHD8) (n = 2), and DNA methyl transferase 1 (DNMT1) (n = 2). In a separate analysis of 347 primary PanNETs, we found loss or deletion of DAXX and ATRX, disruption of SETD2 function (based on loss of H3 lysine 36 trimethylation), loss of ARID1A expression or deletions in CDKN2A in 81% of primary PanNETs with distant metastases. Among patients with loss or deletion of at least 1 of these proteins or genes, 39% survived disease-free for 5 years and 44% had disease-specific survival times of 10 years. Among patients without any of these alterations, 98% survived disease-free for 5 years and 95% had disease-specific survival times of 10 years. Therefore, primary PanNETs with loss of DAXX, ATRX, H3 lysine 36 trimethylation, ARID1A, and/or CDKN2A associate with shorter survival times of patients. Our findings indicate that alterations in chromatin-remodeling genes and CDKN2A contribute to metastasis of PanNETs.


Assuntos
Biomarcadores Tumorais/genética , Inibidor de Quinase Dependente de Ciclina p18/genética , Tumores Neuroendócrinos/genética , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Montagem e Desmontagem da Cromatina/genética , Inibidor p16 de Quinase Dependente de Ciclina , Variações do Número de Cópias de DNA , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mutação , Tumores Neuroendócrinos/mortalidade , Tumores Neuroendócrinos/patologia , Tumores Neuroendócrinos/cirurgia , Pancreatectomia , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/cirurgia , Prognóstico , Sequenciamento Completo do Exoma
16.
EMBO Rep ; 18(6): 861-863, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28500256

RESUMO

The phenomenon of gradual telomere shortening has become a paradigm for how we understand the biology of aging and cancer. Cell proliferation is accompanied by cumulative telomere loss, and the aged cell either senesces, dies or transforms toward cancer. This transformation requires the activation of telomere elongation mechanisms in order to restore telomere length such that cell death or senescence programs are not induced. Most of the time, this occurs through telomerase reactivation. In other rare cases, the Alternative lengthening of telomeres (ALT) pathway hijacks DNA recombination-associated mechanisms to hyperextend telomeres, often to more than 50 kb. Why telomere length is restricted and what sets their maximal length has been a long-standing puzzle in cell biology. Two recent studies published in this issue of EMBO Reports [1] and recently in Science [2] sought to address this important question. Both built on omics approaches that identified ZBTB48 as a potential telomere-associated protein and reveal it to be a critical regulator of telomere length homeostasis by the telomere trimming mechanism. These discoveries provide fundamental insights for our understanding of telomere trimming and how it impacts telomere integrity in stem and cancer cells.


Assuntos
Telomerase/genética , Telômero , Animais , Proteínas de Transporte , Homeostase do Telômero , Proteínas de Ligação a Telômeros , Vertebrados
17.
Clin Cancer Res ; 23(2): 600-609, 2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-27407094

RESUMO

PURPOSE: Pancreatic neuroendocrine tumors (PanNET) are a heterogeneous group of neoplasms with increasing incidence and unpredictable behavior. Whole-exome sequencing has identified recurrent mutations in the genes DAXX and ATRX, which correlate with loss of protein expression and alternative lengthening of telomeres (ALT). Both ALT and DAXX/ATRX loss were initially reported to be associated with a favorable prognosis; however, recent studies suggest the contrary. Our aims were to assess the prevalence and prognostic significance of ALT and DAXX/ATRX in both primary and metastatic PanNETs. EXPERIMENTAL DESIGN: Telomere-specific FISH and DAXX/ATRX IHC was performed on a multi-institutional cohort of 321 patients with resected PanNET and 191 distant metastases from 52 patients. These results were correlated with clinicopathologic features, including disease-free survival (DFS) and disease-specific survival (DSS). RESULTS: The prevalence of ALT and DAXX/ATRX loss in resected PanNETs was 31% and 26%, respectively, and associated with larger tumor size, higher WHO grade, lymph node metastasis, and distant metastasis (P < 0.001). The 5-year DFS and 10-year DSS of patients with ALT-positive and DAXX/ATRX-negative PanNETs were 40% and 50%, respectively, as compared with 96% and 89%, respectively, for wild-type PanNETs. Among distant metastases, ALT and DAXX/ATRX loss was 67% and 52%, respectively, and only occurred in the setting of an ALT-positive and DAXX/ATRX-negative primary PanNET. By multivariate analysis, both ALT and DAXX/ATRX loss were negative, independent prognostic factors for DFS. CONCLUSIONS: ALT and DAXX/ATRX loss in PanNETs was associated with shorter DFS and DSS and likely plays a significant role in driving metastatic disease. Clin Cancer Res; 23(2); 600-9. ©2016 AACR.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma Neuroendócrino/genética , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Homeostase do Telômero/genética , Proteína Nuclear Ligada ao X/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Neuroendócrino/patologia , Proteínas Correpressoras , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Metástase Linfática/genética , Masculino , Pessoa de Meia-Idade , Chaperonas Moleculares , Metástase Neoplásica , Neoplasias Pancreáticas/patologia , Telômero , Sequenciamento Completo do Exoma
18.
J Biol Chem ; 292(6): 2470-2484, 2017 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-28003368

RESUMO

Recent genome-wide studies found that patients with hypotonia, developmental delay, intellectual disability, congenital anomalies, characteristic facial dysmorphic features, and low cholesterol levels suffer from Kaufman oculocerebrofacial syndrome (KOS, also reported as blepharophimosis-ptosis-intellectual disability syndrome). The primary cause of KOS is autosomal recessive mutations in the gene UBE3B However, to date, there are no studies that have determined the cellular or enzymatic function of UBE3B. Here, we report that UBE3B is a mitochondrion-associated protein with homologous to the E6-AP Cterminus (HECT) E3 ubiquitin ligase activity. Mutating the catalytic cysteine (C1036A) or deleting the entire HECT domain (amino acids 758-1068) results in loss of UBE3B's ubiquitylation activity. Knockdown of UBE3B in human cells induces changes in mitochondrial morphology and physiology, a decrease in mitochondrial volume, and a severe suppression of cellular proliferation. We also discovered that UBE3B interacts with calmodulin via its N-terminal isoleucine-glutamine (IQ) motif. Deletion of the IQ motif (amino acids 29-58) results in loss of calmodulin binding and a significant increase in the in vitro ubiquitylation activity of UBE3B. In addition, we found that changes in calcium levels in vitro disrupt the calmodulin-UBE3B interaction. These studies demonstrate that UBE3B is an E3 ubiquitin ligase and reveal that the enzyme is regulated by calmodulin. Furthermore, the modulation of UBE3B via calmodulin and calcium implicates a role for calcium signaling in mitochondrial protein ubiquitylation, protein turnover, and disease.


Assuntos
Calmodulina/metabolismo , Mitocôndrias/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Silenciamento de Genes , Humanos , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética
19.
Cell Rep ; 17(7): 1858-1871, 2016 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-27829156

RESUMO

Cancer cells rely on the activation of telomerase or the alternative lengthening of telomeres (ALT) pathways for telomere maintenance and survival. ALT involves homologous recombination (HR)-dependent exchange and/or HR-associated synthesis of telomeric DNA. Utilizing proximity-dependent biotinylation (BioID), we sought to determine the proteome of telomeres in cancer cells that employ these distinct telomere elongation mechanisms. Our analysis reveals that multiple DNA repair networks converge at ALT telomeres. These include the specialized translesion DNA synthesis (TLS) proteins FANCJ-RAD18-PCNA and, most notably, DNA polymerase eta (Polη). We observe that the depletion of Polη leads to increased ALT activity and late DNA polymerase δ (Polδ)-dependent synthesis of telomeric DNA in mitosis. We propose that Polη fulfills an important role in managing replicative stress at ALT telomeres, maintaining telomere recombination at tolerable levels and stimulating DNA synthesis by Polδ.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Proteômica/métodos , Homeostase do Telômero , Telômero/metabolismo , Biotinilação , DNA/biossíntese , DNA Polimerase III/metabolismo , Replicação do DNA , Células HeLa , Humanos , Mitose , Reparo de DNA por Recombinação
20.
Curr Protoc Cytom ; 73: 12.40.1-12.40.31, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26132175

RESUMO

In cancer cells, telomere length maintenance occurs largely via the direct synthesis of TTAGGG repeats at chromosome ends by telomerase, or less frequently by the recombination-dependent alternative lengthening of telomeres (ALT) pathway. The latter is characterized by the atypical clustering of telomeres within promyelocytic leukemia (PML) nuclear bodies, which harbor proteins that are linked with DNA repair and recombination activity. For this reason, it is speculated that these associated PML bodies represent the sites of the recombination that maintains telomere length. The protocols described here can be employed for the routine investigation of the structural integrity of telomeres and the association of proteins at telomeres in normal cells, challenged cells, and archived formalin-fixed paraffin-embedded clinical tissue specimens that may have activated the ALT pathway.


Assuntos
Imunofluorescência/métodos , Telômero/metabolismo , Sequência de Bases , Adesão Celular , Centrifugação , Cromossomos , DNA/metabolismo , Humanos , Hibridização in Situ Fluorescente , Mitose , Troca de Cromátide Irmã , Bancos de Tecidos
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