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1.
Nat Commun ; 10(1): 2910, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266951

RESUMO

PARP inhibitors (PARPis) have clinical efficacy in BRCA-deficient cancers, but not BRCA-intact tumors, including glioblastoma (GBM). We show that MYC or MYCN amplification in patient-derived glioblastoma stem-like cells (GSCs) generates sensitivity to PARPi via Myc-mediated transcriptional repression of CDK18, while most tumors without amplification are not sensitive. In response to PARPi, CDK18 facilitates ATR activation by interacting with ATR and regulating ATR-Rad9/ATR-ETAA1 interactions; thereby promoting homologous recombination (HR) and PARPi resistance. CDK18 knockdown or ATR inhibition in GSCs suppressed HR and conferred PARPi sensitivity, with ATR inhibitors synergizing with PARPis or sensitizing GSCs. ATR inhibitor VE822 combined with PARPi extended survival of mice bearing GSC-derived orthotopic tumors, irrespective of PARPi-sensitivity. These studies identify a role of CDK18 in ATR-regulated HR. We propose that combined blockade of ATR and PARP is an effective strategy for GBM, even for low-Myc GSCs that do not respond to PARPi alone, and potentially other PARPi-refractory tumors.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Quinases Ciclina-Dependentes/genética , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/metabolismo , Recombinação Homóloga , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Camundongos , Camundongos SCID , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Células-Tronco Neoplásicas/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Gene ; 712: 143954, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31288058

RESUMO

BACKGROUND: Breast cancer (BC) is the highest cause of mortality among female cancer patients. In some cases, BC is due to Poly [ADP-ribose] polymerase 1 (PARP1) gene dysregulation, which has been involved in various important cellular processes. Among Iranian women, the association between PARP1 polymorphisms and BC was never studied before so in this case-control study, the genetic association of three SNPs (rs1136410, rs907187 and rs4653734) was analyzed with susceptibility to BC. METHODS: The study subjects were 386 Iranian females divided into 186 patients and 200 healthy controls. The genotypes of PARP1 variants were detected using ARMS and a combined ARMS-RFLP PCR method. RESULTS: The results showed that Carriers of CG and GG genotypes of the variant rs4653734 were at higher risk of BC compared with wild-type carriers (CC) and this variant was statistically significant under a recessive model of inheritance. Moreover, rs907187 was related to increased BC risk in the CC and GG genotypes under dominant and recessive models of inheritance. The G allele frequency of rs4653734 and rs907187 was higher in breast cancer patients than in normal subjects. No association was detected between rs1136410 and susceptibility to BC among studied groups. Furthermore, A-G-C haplotype was linked to an increased BC risk, whereas A-C-C and A-C-G haplotypes were related to a decreased risk of BC. In Silico predictions suggested that rs907187 affects E2F and E2F-4 transcription factors binding site. CONCLUSIONS: The current study suggests that rs907187 and rs4653734 have remarkable associations with BC risk among Iranian women.


Assuntos
Neoplasias da Mama/genética , Desequilíbrio de Ligação , Poli(ADP-Ribose) Polimerase-1/genética , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Alelos , Neoplasias da Mama/metabolismo , Estudos de Casos e Controles , Biologia Computacional , Ilhas de CpG , Feminino , Regulação Neoplásica da Expressão Gênica , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Haplótipos , Humanos , Irã (Geográfico)/epidemiologia , Pessoa de Meia-Idade , Metástase Neoplásica , Polimorfismo de Fragmento de Restrição , Fatores de Risco , Fatores de Transcrição/metabolismo
3.
Cancer Res ; 79(10): 2460-2461, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31092408

RESUMO

There is extensive literature on PARP1, owing to the multiple functions of PARP1 for DNA repair, aging, chromatin remodeling and transcription, and to the successful clinical development of four PARP inhibitors. In this issue of Cancer Research, Wang and colleagues identified phosphatase 1 nuclear targeting subunit (PNUTS, also referred to as PP1R10) as a partner of PARP1 and demonstrated that PNUTS and PARP1 are interdependent for their recruitment to DNA damage sites. As PARP1 is a prominent target for cancer therapy and PNUTS has PARP-dependent as well as PARP1-independent activities, the findings of Wang and colleagues add insight into DNA repair mechanisms with potential application to drug targeting and precision therapy.See related article by Wang et al., p. 2526.


Assuntos
Reparo do DNA/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases , Dano ao DNA/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/genética , Poli(ADP-Ribose) Polimerase-1/genética
4.
Nat Commun ; 10(1): 1635, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967556

RESUMO

Chordomas are rare bone tumors with few therapeutic options. Here we show, using whole-exome and genome sequencing within a precision oncology program, that advanced chordomas (n = 11) may be characterized by genomic patterns indicative of defective homologous recombination (HR) DNA repair and alterations affecting HR-related genes, including, for example, deletions and pathogenic germline variants of BRCA2, NBN, and CHEK2. A mutational signature associated with HR deficiency was significantly enriched in 72.7% of samples and co-occurred with genomic instability. The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib, which is preferentially toxic to HR-incompetent cells, led to prolonged clinical benefit in a patient with refractory chordoma, and whole-genome analysis at progression revealed a PARP1 p.T910A mutation predicted to disrupt the autoinhibitory PARP1 helical domain. These findings uncover a therapeutic opportunity in chordoma that warrants further exploration, and provide insight into the mechanisms underlying PARP inhibitor resistance.


Assuntos
Cordoma/tratamento farmacológico , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Poli(ADP-Ribose) Polimerase-1/genética , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Reparo de DNA por Recombinação/genética , Adulto , Idoso , Cordoma/genética , Cordoma/patologia , Mapeamento Cromossômico , Quebras de DNA de Cadeia Dupla , Análise Mutacional de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Instabilidade Genômica , Humanos , Masculino , Pessoa de Meia-Idade , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Medicina de Precisão/métodos , Domínios Proteicos/genética , Resultado do Tratamento , Sequenciamento Completo do Exoma
5.
Oncol Rep ; 41(6): 3555-3564, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31002368

RESUMO

Neoplastic transformation is characterized by metabolic rewiring to sustain the elevated biosynthetic demands of highly proliferative cancer cells. To obtain the precursors for macromolecule biosynthesis, cancer cells avidly uptake and metabolize glucose and glutamine. Thus, targeting the availability or metabolism of these nutrients is an attractive anticancer therapeutic strategy. To improve our knowledge concerning how cancer cells respond to nutrient withdrawal, the response to glutamine and/or glucose starvation was studied in human in vitro transformed fibroblasts, deeply characterized at the cellular and molecular level. Concomitant starvation of both nutrients led to rapid loss of cellular adhesion (~16 h after starvation), followed by cell death. Deprivation of glucose alone had the same effect, although at a later time (~48 h after starvation), suggesting that glucose plays a key role in enabling cell attachment to the extracellular matrix. Glutamine deprivation did not induce rapid cell death, but caused a prolonged arrest of cellular proliferation; the cells started dying only 96 h after starvation. Before massive cell death occurred, the effects of all the starvation conditions were reversible. Autophagy activation was observed in cells incubated in the absence of glucose for more than 48 h, while autophagy was not detected under the other starvation conditions. Markers of apoptotic cell death, such as caspase 3, caspase 9 and poly(ADP­ribose) polymerase 1 (PARP­1) proteolytic fragments, were not observed under any growth condition. Glucose and/or glutamine deprivation caused very rapid PARP­1 activation, with marked PARP­1 (poly­ADP) ribosylation and protein (poly­ADP) ribosylation. This activation was not due to starvation­induced DNA double­strand breaks, which appeared at the late stages of deprivation, when most cells died. Collectively, these results highlight a broad range of consequences of glucose and glutamine starvation, which may be taken into account when nutrient availability is used as a target for anticancer therapies.


Assuntos
Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Glucose/metabolismo , Glutamina/metabolismo , Apoptose/genética , Autofagia/genética , Caspases/genética , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Morte Celular/genética , Transformação Celular Neoplásica/metabolismo , Quebras de DNA de Cadeia Dupla , Fibroblastos/metabolismo , Fibroblastos/patologia , Glucose/genética , Glutamina/genética , Humanos , Terapia de Alvo Molecular , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inanição/genética , Inanição/metabolismo
6.
Nat Commun ; 10(1): 1307, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30898999

RESUMO

Atrial fibrillation (AF) is the most common clinical tachyarrhythmia with a strong tendency to progress in time. AF progression is driven by derailment of protein homeostasis, which ultimately causes contractile dysfunction of the atria. Here we report that tachypacing-induced functional loss of atrial cardiomyocytes is precipitated by excessive poly(ADP)-ribose polymerase 1 (PARP1) activation in response to oxidative DNA damage. PARP1-mediated synthesis of ADP-ribose chains in turn depletes nicotinamide adenine dinucleotide (NAD+), induces further DNA damage and contractile dysfunction. Accordingly, NAD+ replenishment or PARP1 depletion precludes functional loss. Moreover, inhibition of PARP1 protects against tachypacing-induced NAD+ depletion, oxidative stress, DNA damage and contractile dysfunction in atrial cardiomyocytes and Drosophila. Consistently, cardiomyocytes of persistent AF patients show significant DNA damage, which correlates with PARP1 activity. The findings uncover a mechanism by which tachypacing impairs cardiomyocyte function and implicates PARP1 as a possible therapeutic target that may preserve cardiomyocyte function in clinical AF.


Assuntos
Fibrilação Atrial/metabolismo , Fibrilação Atrial/prevenção & controle , Modelos Cardiovasculares , Miócitos Cardíacos/enzimologia , NAD/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Animais , Fibrilação Atrial/genética , Fibrilação Atrial/fisiopatologia , Benzimidazóis/farmacologia , Células Cultivadas , Dano ao DNA , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/metabolismo , Ativação Enzimática/efeitos dos fármacos , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/enzimologia , Átrios do Coração/fisiopatologia , Humanos , Larva/efeitos dos fármacos , Larva/metabolismo , Camundongos , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologia , Miócitos Cardíacos/patologia , Niacinamida/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Marca-Passo Artificial/efeitos adversos , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Pupa/efeitos dos fármacos , Pupa/metabolismo , Ratos , Ratos Wistar
7.
Nat Commun ; 10(1): 1203, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867423

RESUMO

Vascular calcification is highly prevalent in end-stage renal diseases and is predictive of cardiovascular events and mortality. Poly(ADP-ribose) polymerase 1 (PARP1) inhibition or deletion is vasoprotective in several disease models. Here we show that PARP activity is increased in radial artery samples from patients with chronic renal failure, in arteries from uraemic rats, and in calcified vascular smooth muscle cells (VSMCs) in vitro. PARP1 deficiency blocks, whereas PARP1 overexpression exacerbates, the transdifferentiation of VSMCs from a contractile to an osteogenic phenotype, the expression of mineralization-regulating proteins, and calcium deposition. PARP1 promotes Runx2 expression, and Runx2 deficiency offsets the pro-calcifying effects of PARP1. Activated PARP1 suppresses miRNA-204 expression via the IL-6/STAT3 pathway and thus relieves the repression of its target, Runx2, resulting in increased Runx2 protein. Together, these results suggest that PARP1 counteracts vascular calcification and that therapeutic agents that influence PARP1 activity may be of benefit to treat vascular calcification.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Falência Renal Crônica/complicações , MicroRNAs/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Calcificação Vascular/patologia , Adenina/toxicidade , Animais , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Interleucina-6/metabolismo , Masculino , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Artéria Radial/patologia , Ratos , Ratos Wistar , Fator de Transcrição STAT3/metabolismo , Técnicas de Cultura de Tecidos , Regulação para Cima , Uremia/induzido quimicamente , Uremia/complicações , Calcificação Vascular/etiologia
8.
Ann Hematol ; 98(6): 1383-1392, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30877373

RESUMO

Poly (ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that participates in the DNA repair of malignant cells, with various consequences on their survival. We have recently shown that PARP1 mRNA levels in the bone marrow of patients with myelodysplastic syndrome (MDS) are correlated to prognosis. To evaluate PARP1 as a biomarker of response to 5-azacytidine in patients with MDS, we measured PARP1 mRNA levels by a quantitative real-time PCR in diagnostic bone marrow samples of 77 patients with MDS treated with 5-azacytidine. Patients with higher PARP1 mRNA levels had a better response to 5-azacytidine per the IWG criteria (p = 0.006) and a longer median survival after 5-azacytidine initiation (p = 0.033). Multivariate analysis revealed that PARP1 mRNA level was the only factor affecting response to treatment and survival after treatment with 5-azacytidine. A next-generation sequencing for 40 genes of interest in MDS and quantification of the methylation levels of the PARP1 promoter were also carried out in a subset of samples (16 and 18 samples respectively). It is the first time that a single, easily measurable biomarker shows a clear correlation with response to treatment and survival in a patient population consisting of previously untreated patients with MDS homogeneously treated with 5-azacytidine. The fact that PARP1 is also a treatment target in several malignancies underscores the importance of our finding for the potential use of PARP1 inhibitors in MDS.


Assuntos
Antimetabólitos/uso terapêutico , Azacitidina/uso terapêutico , Medula Óssea/química , Síndromes Mielodisplásicas/tratamento farmacológico , Poli(ADP-Ribose) Polimerase-1/biossíntese , RNA Mensageiro/análise , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos/efeitos adversos , Azacitidina/efeitos adversos , Biomarcadores , Dano ao DNA , Metilação de DNA/efeitos dos fármacos , Reparo do DNA , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Prognóstico , Regiões Promotoras Genéticas/efeitos dos fármacos , Modelos de Riscos Proporcionais , Regulação para Cima/efeitos dos fármacos
9.
Virus Res ; 265: 104-114, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30910697

RESUMO

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease and severe neural complications in infants and young children. Viral pathogenesis is associated with virus-induced cell death and inflammatory cytokine production, which is usually correlated with the type of programmed cell death. EV71-infected cells were analyzed through microscopy, cell staining, and immunoblotting to determine the characteristics of EV71-induced cell death. Results demonstrated that EV71 infection induced cell shrinkage, nuclear condensation, decreased mitochondrial potential, and membrane phosphatidylserine translocation. Caspase-9 activation, poly (ADP-ribose) polymerase cleavage, and lactate dehydrogenase release were also observed during virus-induced cell death. The activated gasdermin D (GSDMD) and the phosphorylated mixed lineage kinase domain-like protein (p-MLKL) were not detected. These observations indicated that EV71-induced cell death was mainly executed by apoptosis through the intrinsic pathway rather than by GSDMD-mediated pyroptosis and p-MLKL-mediated necroptosis. Genome scanning analysis identified that EV71 2A, 2B, and 3C might be the determinant genes of virus-induced cell death. Further experiments showed that EV71 2A- and 3C-induced cell death exhibited dependence on their protease activities but involved different mechanisms. EV71 2A-induced cell death was correlated with the shut-off of host cap-dependent translation, whereas EV71 3C-induced cell death might not be ascribed to this mechanism. These findings would enhance our understanding of EV71 infection and viral pathogenesis, and help identify antiviral targets.


Assuntos
Apoptose/genética , Morte Celular/genética , Enterovirus Humano A/genética , Enterovirus Humano A/patogenicidade , Genes Virais , Caspase 9/genética , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Poli(ADP-Ribose) Polimerase-1/genética , Proteínas Virais/genética
10.
Epigenetics Chromatin ; 12(1): 15, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30777121

RESUMO

BACKGROUND: Recently, we showed that PARP1 is involved in cotranscriptional splicing, possibly by bridging chromatin to RNA and recruiting splicing factors. It also can influence alternative splicing decisions through the regulation of RNAPII elongation. In this study, we investigated the effect of PARP1-mediated chromatin changes on RNAPII movement, during transcription and alternative splicing. RESULTS: We show that RNAPII pauses at PARP1-chromatin structures within the gene body. Knockdown of PARP1 abolishes this RNAPII pausing, suggesting that PARP1 may regulate RNAPII elongation. Additionally, PARP1 alters nucleosome deposition and histone post-translational modifications at specific exon-intron boundaries, thereby affecting RNAPII movement. Lastly, genome-wide analyses confirmed that PARP1 influences changes in RNAPII elongation by either reducing or increasing the rate of RNAPII elongation depending on the chromatin context. CONCLUSIONS: These studies suggest a context-specific effect of PARP1-chromatin binding on RNA polymerase movement and provide a platform to delineate PARP1's role in RNA biogenesis and processing.


Assuntos
Montagem e Desmontagem da Cromatina , Processamento de RNA , Elongação da Transcrição Genética , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , RNA Polimerase II/metabolismo
11.
Int J Cancer ; 145(2): 474-483, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-30614530

RESUMO

The poly(ADP-ribose) polymerases (PARP) play important roles in repairing damaged DNA during intrinsic cell death. We recently linked PARP-1 to death receptor (DR)-activated extrinsic apoptosis, the present studies sought to elucidate the function of cytoplasmic PARP-1 in pancreatic cancer tumorigenesis and therapy. Using human normal and pancreatic cancer tissues, we analyzed the prevalence of cytoplasmic PARP-1 expression. In normal human pancreatic tissues, PARP-1 expression was present in the nucleus; however, cytoplasmic PARP-1 expression was identified in pancreatic cancers. Therefore, cytoplasmic PARP-1 mutants were generated by site-direct mutagenesis, to determine a causative effect of cytoplasmic PARP-1 on pancreatic cancer tumorigenesis and sensitivity to therapy with TRA-8, a humanized DR5 antibody. PARP-1 cytoplasmic mutants rendered TRA-8 sensitive pancreatic cancer cells, BxPc-3 and MiaPaCa-2, more resistant to TRA-8-induced apoptosis; whereas wild-type PARP-1, localizing mainly in the nucleus, had no effects. Additionally, cytoplasmic PARP-1, but not wild-type PARP-1, increased resistance of BxPc-3 cells to TRA-8 therapy in a mouse xenograft model in vivo. Inhibition of PARP enzymatic activity attenuated cytoplasmic PARP-1-mediated TRA-8 resistance. Furthermore, increased cytoplasmic PARP-1, but not wild-type PARP-1, was recruited into the TRA-8-activated death-inducing signaling complex and associated with increased and sustained activation of Src-mediated survival signals. In contrast, PARP-1 knockdown inhibited Src activation. Taken together, we have identified a novel function and mechanism underlying cytoplasmic PARP-1, distinct from nuclear PARP-1, in regulating DR5-activated apoptosis. Our studies support an innovative application of available PARP inhibitors or new cytoplasmic PARP-1 antagonists to enhance TRAIL therapy for TRAIL-resistant pancreatic cancers.


Assuntos
Citoplasma/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pancreáticas/patologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Mutagênese Sítio-Dirigida , Gradação de Tumores , Transplante de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fenantrenos/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
12.
Int J Mol Sci ; 20(3)2019 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-30691122

RESUMO

Poly- adenosine diphosphate (ADP)-ribose (PAR) is a polymer synthesized as a posttranslational modification by some poly (ADP-ribose) polymerases (PARPs), namely PARP-1, PARP-2, tankyrase-1, and tankyrase-2 (TNKS-1/2). PARP-1 is nuclear and has also been detected in extracellular vesicles. PARP-2 and TNKS-1/2 are distributed in nuclei and cytoplasm. PARP or PAR alterations have been described in tumors, and in particular by influencing the Epithelial- Mesenchymal Transition (EMT), which influences cell migration and drug resistance in cancer cells. Pro-EMT and anti-EMT effects of PARP-1 have been reported while whether PAR changes occur specifically during EMT is currently unknown. The PARP-1/2 inhibitor Olaparib (OLA) is approved by FDA to treat certain patients harboring cancers with impaired homologous recombination. Here, we studied PAR changes and OLA effects on EMT. Total and nuclear PAR increased in EMT while PAR belts were disassembled. OLA prevented EMT, according to: (i) molecular markers evaluated by immuno-cytofluorescence/image quantification, Western blots, and RNA quantitation, (ii) morphological changes expressed as anisotropy, and (iii) migration capacity in the scratch assay. OLA also partially reversed EMT. OLA might work through unconventional mechanisms of action (different from synthetic lethality), even in non-BRCA (breast cancer 1 gene) mutated cancers.


Assuntos
Glândulas Mamárias Animais/citologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerases/genética , Fator de Crescimento Transformador beta/efeitos adversos , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulação para Baixo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Camundongos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo
13.
J Biol Chem ; 294(8): 2827-2838, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30598506

RESUMO

Ribosomal proteins are the building blocks of ribosome biogenesis. Beyond their known participation in ribosome assembly, the ribosome-independent functions of ribosomal proteins are largely unknown. Here, using immunoprecipitation, subcellular fractionation, His-ubiquitin pulldown, and immunofluorescence microscopy assays, along with siRNA-based knockdown approaches, we demonstrate that ribosomal protein L6 (RPL6) directly interacts with histone H2A and is involved in the DNA damage response (DDR). We found that in response to DNA damage, RPL6 is recruited to DNA damage sites in a poly(ADP-ribose) polymerase (PARP)-dependent manner, promoting its interaction with H2A. We also observed that RPL6 depletion attenuates the interaction between mediator of DNA damage checkpoint 1 (MDC1) and H2A histone family member X, phosphorylated (γH2AX), impairs the accumulation of MDC1 at DNA damage sites, and reduces both the recruitment of ring finger protein 168 (RNF168) and H2A Lys-15 ubiquitination (H2AK15ub). These RPL6 depletion-induced events subsequently inhibited the recruitment of the following downstream repair proteins: tumor protein P53-binding protein 1 (TP53BP1) and BRCA1, DNA repair-associated (BRCA1). Moreover, the RPL6 knockdown resulted in defects in the DNA damage-induced G2-M checkpoint, DNA damage repair, and cell survival. In conclusion, our study identifies RPL6 as a critical regulatory factor involved in the DDR. These findings expand our knowledge of the extraribosomal functions of ribosomal proteins in cell physiology and deepen our understanding of the molecular mechanisms underlying DDR regulation.


Assuntos
Proteína BRCA1/metabolismo , Dano ao DNA , Reparo do DNA , Histonas/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas Ribossômicas/metabolismo , Proteína BRCA1/genética , Ciclo Celular , Sobrevivência Celular , Células HEK293 , Células HeLa , Histonas/genética , Humanos , Poli(ADP-Ribose) Polimerase-1/genética , Proteínas Ribossômicas/genética , Transdução de Sinais , Ubiquitina , Ubiquitinação
14.
EBioMedicine ; 40: 375-381, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30635165

RESUMO

BACKGROUND: Locally advanced pancreatic cancer (LAPC) has a dismal prognosis with current treatment modalities and one-third of patients die from local progression of disease. Preclinical studies with orthotopic PC demonstrated dramatic synergy between radiotherapy (RT) and the poly(ADP-ribose) polymerase-1/2 inhibitor (PARPi), veliparib. We conducted a phase I trial of gemcitabine, radiotherapy and dose-escalated veliparib in LAPC. METHODS: This was a single institution investigator-initiated open-label, single-arm phase 1 clinical trial (NCT01908478). Weekly gemcitabine with daily IMRT and veliparib dose escalated using a Bayesian adaptive design were administered in treatment naïve LA or borderline resectable PC. The primary end point was identification of the MTD. Secondary endpoints included efficacy, characterization of PAR levels using ELISA, DDR alterations with targeted next generation sequencing and transcriptome analysis, tumor mutation burden (TMB) and microsatellite instability (MSI) status. FINDINGS: Thirty patients were enrolled. The MTD of veliparib was 40 mg BID with gemcitabine 400 mg/m2 and RT (36 Gy/15 fractions). Sixteen DLTs were identified in 12 patients. Grade ≥ 3 adverse events included lymphopenia (96%) and anemia (36%). Median OS for all patients was 15 months. Median OS for DDR pathway gene altered and intact cases was 19 months (95% CI: 6.2-27.2) and 14 months (95% CI: 10.0-21.8), respectively. There were no significant associations between levels of PAR, TMB, or MSI with outcomes. The DDR transcripts PARP3 and RBX1 significantly correlated with OS. INTERPRETATION: This is the first report of a PARPi-chemoradiotherapy combination in PC. The regimen was safe, tolerable at the RP2D, and clinically active as an upfront treatment strategy in patients biologically unselected by upfront chemotherapy. Expression of the DDR transcripts, PARP3 and RBX1, were associated with OS suggesting validation in a follow up phase 2 study. FUND: Phase One Foundation; National Institutes of Health [1R01CA188480-01A1, P01 CA098912]. Veliparib was provided by Abbvie.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Radioterapia , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Benzimidazóis/administração & dosagem , Terapia Combinada , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Feminino , Humanos , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Mutação , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias Pancreáticas/mortalidade , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Poli(ADP-Ribose) Polimerases , Prognóstico , Radioterapia/métodos , Resultado do Tratamento
15.
Gynecol Oncol ; 153(1): 127-134, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30686551

RESUMO

OBJECTIVE: Poly(ADP-ribose) polymerase (PARP) inhibitors have shown substantial activity in homologous recombination- (HR-) deficient ovarian cancer and are undergoing testing in other HR-deficient tumors. For reasons that are incompletely understood, not all patients with HR-deficient cancers respond to these agents. Preclinical studies have demonstrated that changes in alternative DNA repair pathways affect PARP inhibitor (PARPi) sensitivity in ovarian cancer models. This has not previously been assessed in the clinical setting. METHODS: Clonogenic and plasmid-based HR repair assays were performed to compare BRCA1-mutant COV362 ovarian cancer cells with or without 53BP1 gene deletion. Archival biopsies from ovarian cancer patients in the phase I, open-label clinical trial of PARPi ABT-767 were stained for PARP1, RAD51, 53BP1 and multiple components of the nonhomologous end-joining (NHEJ) DNA repair pathway. Modified histochemistry- (H-) scores were determined for each repair protein in each sample. HRD score was determined from tumor DNA. RESULTS: 53BP1 deletion increased HR in BRCA1-mutant COV362 cells and decreased PARPi sensitivity in vitro. In 36 women with relapsed ovarian cancer, responses to the PARPi ABT-767 were observed exclusively in cancers with HR deficiency. In this subset, 7 of 18 patients (39%) had objective responses. The actual HRD score did not further correlate with change from baseline tumor volume (r = 0.050; p = 0.87). However, in the HR-deficient subset, decreased 53BP1 H-score was associated with decreased antitumor efficacy of ABT-767 (r = -0.69, p = 0.004). CONCLUSION: Differences in complementary repair pathways, particularly 53BP1, correlate with PARPi response of HR-deficient ovarian cancers.


Assuntos
Benzamidas/administração & dosagem , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Sulfonamidas/administração & dosagem , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Reparo do DNA , Resistencia a Medicamentos Antineoplásicos , Feminino , Genes BRCA1 , Genes BRCA2 , Recombinação Homóloga , Humanos , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/biossíntese , Poli(ADP-Ribose) Polimerase-1/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/biossíntese , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência
16.
Mol Genet Genomic Med ; 7(3): e556, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30680959

RESUMO

BACKGROUND: Identification of genetic factors causing predisposition to renal cell carcinoma has helped improve screening, early detection, and patient survival. METHODS: We report the characterization of a proband with renal and thyroid cancers and a family history of renal and other cancers by whole-exome sequencing (WES), coupled with WES analysis of germline DNA from additional affected and unaffected family members. RESULTS: This work identified multiple predicted protein-damaging variants relevant to the pattern of inherited cancer risk. Among these, the proband and an affected brother each had a heterozygous Ala45Thr variant in SDHA, a component of the succinate dehydrogenase (SDH) complex. SDH defects are associated with mitochondrial disorders and risk for various cancers; immunochemical analysis indicated loss of SDHB protein expression in the patient's tumor, compatible with SDH deficiency. Integrated analysis of public databases and structural predictions indicated that the two affected individuals also had additional variants in genes including TGFB2, TRAP1, PARP1, and EGF, each potentially relevant to cancer risk alone or in conjunction with the SDHA variant. In addition, allelic imbalances of PARP1 and TGFB2 were detected in the tumor of the proband. CONCLUSION: Together, these data suggest the possibility of risk associated with interaction of two or more variants.


Assuntos
Carcinoma de Células Renais/genética , Mutação em Linhagem Germinativa , Neoplasias Renais/genética , Adulto , Idoso , Complexo II de Transporte de Elétrons/genética , Epistasia Genética , Feminino , Proteínas de Choque Térmico HSP90/genética , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Poli(ADP-Ribose) Polimerase-1/genética , Fator de Crescimento Transformador beta2/genética
17.
Oncogene ; 38(15): 2750-2766, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30542118

RESUMO

Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification by which poly ADP-ribose (PAR) polymers are covalently added to proteins through a PAR polymerase (PARP). Here, using proteomic approach, we identify the transcriptional regulator, OVOL2, is a novel substrate of PARP1 and can be PARylated at residues Lysine 145, Lysine 176, and Lysine 212 within its C2H2 zinc finger domains. Overexpression of PARylated OVOL2 alters cell morphology and induces lagging chromosomes and aneuploidy. To define the underlying molecular mechanism by which OVOL2 induces abnormal cell cycle and centrosome amplification, we uncover that the OVOL2 elevates the protein levels of Cyclin E by enhancing its stability. Furthermore, we identify Skp2, the E3 ubiquitin ligase of Cyclin E, as a direct target of PARylated OVOL2. Using ChIP assay, the OVOL2 binding site on the promoter region of Skp2 is mapped. To further explore the physiological effect, we show that PARylated OVOL2 can induce cell death. Furthermore, to investigate PARylated OVOL2 function in vivo, we further develop a null-mice xenograft model and generate MMTV-PyVT transgenic mice and monitor the effect of wild-type OVOL2 and non-PARylated OVOL2-3K/A mutants on tumor progression. Consistently, overexpression of wild-type OVOL2 in both null-mice xenograft and MMTV-PyVT transgenic mice displays significantly reduction of tumor progression, respectively, further indicating that the function of OVOL2 as a tumor suppressor in vivo is highly regulated by PARylation. Taken together, our study sheds new light on PARP1-induced PARylation as a critical event in the OVOL2-mediated regulation of chromosomal integrity and suppression of cancer cells growth.


Assuntos
Morte Celular/genética , Poli ADP Ribosilação/genética , Poli Adenosina Difosfato Ribose/genética , Fatores de Transcrição/genética , Aneuploidia , Animais , Sítios de Ligação/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Centrossomo/fisiologia , Cromossomos/genética , Ciclina E/genética , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Transgênicos , Poli(ADP-Ribose) Polimerase-1/genética , Regiões Promotoras Genéticas/genética , Proteômica/métodos , Proteínas Quinases Associadas a Fase S/genética , Transcrição Genética/genética , Ubiquitina-Proteína Ligases/genética
18.
Nucleic Acids Res ; 47(3): 1564-1572, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30551210

RESUMO

Poly (ADP-ribose) polymerase 1 (PARP1) has emerged as an attractive target for cancer therapy due to its key role in DNA repair processes. Inhibition of PARP1 in BRCA-mutated cancers has been observed to be clinically beneficial. Recent genome-mapping experiments have identified a non-canonical G-quadruplex-forming sequence containing bulges within the PARP1 promoter. Structural features, like bulges, provide opportunities for selective chemical targeting of the non-canonical G-quadruplex structure within the PARP1 promoter, which could serve as an alternative therapeutic approach for the regulation of PARP1 expression. Here we report the G-quadruplex structure formed by a 23-nucleotide G-rich sequence in the PARP1 promoter. Our study revealed a three-layered intramolecular (3+1) hybrid G-quadruplex scaffold, in which three strands are oriented in one direction and the fourth in the opposite direction. This structure exhibits unique structural features such as an adenine bulge and a G·G·T base triple capping structure formed between the central edgewise loop, propeller loop and 5' flanking terminal. Given the highly important role of PARP1 in DNA repair and cancer intervention, this structure presents an attractive opportunity to explore the therapeutic potential of PARP1 inhibition via G-quadruplex DNA targeting.


Assuntos
DNA/química , Quadruplex G , Conformação de Ácido Nucleico , Poli(ADP-Ribose) Polimerase-1/química , Adenina/química , DNA/genética , Reparo do DNA/genética , Guanina/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Poli(ADP-Ribose) Polimerase-1/genética , Regiões Promotoras Genéticas
19.
Proc Natl Acad Sci U S A ; 115(40): 10076-10081, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30213852

RESUMO

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classic and alternative nonhomologous end-joining (NHEJ) factors. We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. We show here that in contrast to classic (c-NHEJ) factors, Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class-switch recombination in primary B cells, and inversions in tail fibroblasts that generate Eml4-Alk fusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing of Eml4-Alk junctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting that Parp1 may suppress double-strand break processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.


Assuntos
Linfócitos B/metabolismo , Reparo do DNA por Junção de Extremidades/fisiologia , Switching de Imunoglobulina/fisiologia , Células-Tronco Embrionárias Murinas/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Quinase do Linfoma Anaplásico , Animais , Fibroblastos/metabolismo , Camundongos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo
20.
Oncol Rep ; 40(4): 2381-2388, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30066930

RESUMO

Breast cancer is one of the major health issues confronting women; however, treatment with conventional chemotherapeutic drugs is limited. Currently, paclitaxel is used as a therapeutic clinical agent to treat breast cancer that exerts antitumor activity in numerous types of cancer cell. Curcumin (diferuloylmethane), a polyphenol derived from turmeric (Curcuma longa), possesses several properties that could enable it to exert an anticancer effect. Previous reports have demonstrated the synergistic effects of several chemotherapeutic drugs in combination with curcumin. Therefore, the aim of the current study was to evaluate cell death induced by curcumin and paclitaxel alone and in combination in human breast cancer cell lines: MCF7, an epithelial and luminal-like adenocarcinoma cell line triple positive for estrogen and progesterone receptor, and MDA-MB-234, a metastatic human breast cancer cell line triple negative for such receptors, as well as MCF-10F as a normal breast cell line. The results indicated that curcumin and paclitaxel induced apoptosis and necrosis, which was demonstrated through multiple methods, including assays of caspase-3/7 activity, Annexin V, poly(ADP-ribose) polymerase-1 activation and protein expression of caspase-3, nuclear factor (NF)-κB transcription factor and proliferating cell nuclear antigen. The results identified that the combination of curcumin and paclitaxel had a decreased effect on apoptosis in the malignant MDA-MB-231 cell line compared with in MCF7 or MCF-10F. It was demonstrated that the combined treatment with curcumin and paclitaxel resulted in a higher level of apoptosis compared with either substance alone in breast cancer cell lines. Therefore, breast cancer treatment may benefit from the use of a combination of drugs in chemotherapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Sinergismo Farmacológico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Proliferação de Células/efeitos dos fármacos , Curcumina/administração & dosagem , Feminino , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Paclitaxel/administração & dosagem , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Células Tumorais Cultivadas
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