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1.
Nat Commun ; 11(1): 5239, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067475

RESUMO

The alternative non-homologous end-joining (NHEJ) pathway promotes DNA double-strand break (DSB) repair in cells deficient for NHEJ or homologous recombination, suggesting that it operates at all stages of the cell cycle. Here, we use an approach in which DNA breaks can be induced in G1 cells and their repair tracked, enabling us to show that joining of DSBs is not functional in G1-arrested XRCC4-deficient cells. Cell cycle entry into S-G2/M restores DSB repair by Pol θ-dependent and PARP1-independent alternative NHEJ with repair products bearing kilo-base long DNA end resection, micro-homologies and chromosome translocations. We identify a synthetic lethal interaction between XRCC4 and Pol θ under conditions of G1 DSBs, associated with accumulation of unresolved DNA ends in S-G2/M. Collectively, our results support the conclusion that the repair of G1 DSBs progressing to S-G2/M by alternative NHEJ drives genomic instability and represent an attractive target for future DNA repair-based cancer therapies.


Assuntos
Ciclo Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fase G1 , Camundongos , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo
2.
Nucleic Acids Res ; 48(17): 9694-9709, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32890402

RESUMO

DNA breaks recruit and activate PARP1/2, which deposit poly-ADP-ribose (PAR) to recruit XRCC1-Ligase3 and other repair factors to promote DNA repair. Clinical PARP inhibitors (PARPi) extend the lifetime of damage-induced PARP1/2 foci, referred to as 'trapping'. To understand the molecular nature of 'trapping' in cells, we employed quantitative live-cell imaging and fluorescence recovery after photo-bleaching. Unexpectedly, we found that PARP1 exchanges rapidly at DNA damage sites even in the presence of clinical PARPi, suggesting the persistent foci are not caused by physical stalling. Loss of Xrcc1, a major downstream effector of PAR, also caused persistent PARP1 foci without affecting PARP1 exchange. Thus, we propose that the persistent PARP1 foci are formed by different PARP1 molecules that are continuously recruited to and exchanging at DNA lesions due to attenuated XRCC1-LIG3 recruitment and delayed DNA repair. Moreover, mutation analyses of the NAD+ interacting residues of PARP1 showed that PARP1 can be physically trapped at DNA damage sites, and identified H862 as a potential regulator for PARP1 exchange. PARP1-H862D, but not PARylation-deficient PARP1-E988K, formed stable PARP1 foci upon activation. Together, these findings uncovered the nature of persistent PARP1 foci and identified NAD+ interacting residues involved in the PARP1 exchange.


Assuntos
Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Sítios de Ligação , Domínio Catalítico , Linhagem Celular Tumoral , Reparo do DNA/fisiologia , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Indazóis/farmacologia , Cinética , Imagem Molecular , NAD/metabolismo , Piperidinas/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo
3.
Mol Cell ; 79(6): 934-949.e14, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32822587

RESUMO

Although ADP-ribosylation of histones by PARP-1 has been linked to genotoxic stress responses, its role in physiological processes and gene expression has remained elusive. We found that NAD+-dependent ADP-ribosylation of histone H2B-Glu35 by small nucleolar RNA (snoRNA)-activated PARP-1 inhibits AMP kinase-mediated phosphorylation of adjacent H2B-Ser36, which is required for the proadipogenic gene expression program. The activity of PARP-1 on H2B requires NMNAT-1, a nuclear NAD+ synthase, which directs PARP-1 catalytic activity to Glu and Asp residues. ADP-ribosylation of Glu35 and the subsequent reduction of H2B-Ser36 phosphorylation inhibits the differentiation of adipocyte precursors in cultured cells. Parp1 knockout in preadipocytes in a mouse lineage-tracing genetic model increases adipogenesis, leading to obesity. Collectively, our results demonstrate a functional interplay between H2B-Glu35 ADP-ribosylation and H2B-Ser36 phosphorylation that controls adipogenesis.


Assuntos
ADP-Ribosilação/genética , Adipogenia/genética , Histonas/genética , Poli(ADP-Ribose) Polimerase-1/genética , Adenosina Difosfato Ribose/genética , Adipócitos/metabolismo , Adipócitos/patologia , Animais , Linhagem Celular , Dano ao DNA/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Fosforilação/genética , RNA Nucleolar Pequeno/genética
4.
Nat Commun ; 11(1): 4077, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796846

RESUMO

Double-strand breaks (DSBs) are the most toxic type of DNA lesions. Cells repair these lesions using either end protection- or end resection-coupled mechanisms. To study DSB repair choice, we present the Color Assay Tracing-Repair (CAT-R) to simultaneously quantify DSB repair via end protection and end resection pathways. CAT-R introduces DSBs using CRISPR/Cas9 in a tandem fluorescent reporter, whose repair distinguishes small insertions/deletions from large deletions. We demonstrate CAT-R applications in chemical and genetic screens. First, we evaluate 21 compounds currently in clinical trials which target the DNA damage response. Second, we examine how 417 factors involved in DNA damage response influence the choice between end protection and end resection. Finally, we show that impairing nucleotide excision repair favors error-free repair, providing an alternative way for improving CRISPR/Cas9-based knock-ins. CAT-R is a high-throughput, versatile assay to assess DSB repair choice, which facilitates comprehensive studies of DNA repair and drug efficiency testing.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas Mutadas de Ataxia Telangiectasia/genética , Ciclo Celular , Sobrevivência Celular , Dano ao DNA , Reparo do DNA por Junção de Extremidades , Avaliação Pré-Clínica de Medicamentos , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Poli(ADP-Ribose) Polimerase-1/genética
5.
Nat Commun ; 11(1): 2174, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358582

RESUMO

Poly-ADP-ribosylation (PARylation) is a fully reversible post-translational modification with key roles in cellular physiology. Due to the multi-domain structure of poly(ADP-ribose) polymerase-1 (PARP1) and the highly dynamic nature of the PARylation reaction, studies on the biochemical mechanism and structural dynamics remain challenging. Here, we report label-free, time-resolved monitoring of PARP1-dependent PARylation using ATR-FTIR spectroscopy. This includes PARP1 activation by binding to DNA strand break models, NAD+ substrate binding, PAR formation, and dissociation of automodified PARP1 from DNA. Analyses of PARP1 activation at different DNA models demonstrate a strong positive correlation of PARylation and PARP1 dissociation, with the strongest effects observed for DNA nicks and 3' phosphorylated ends. Moreover, by examining dynamic structural changes of PARP1, we reveal changes in the secondary structure of PARP1 induced by NAD+ and PARP inhibitor binding. In summary, this approach enables holistic and dynamic insights into PARP1-dependent PARylation with molecular and temporal resolution.


Assuntos
Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação/genética , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Humanos , Cinética , NAD/análogos & derivados , NAD/biossíntese , NAD/metabolismo , Oligonucleotídeos/química , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , Poli ADP Ribosilação/efeitos dos fármacos , Poli ADP Ribosilação/fisiologia , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier/métodos
6.
Pol J Pathol ; 71(1): 69-74, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32429658

RESUMO

Molecular next gene sequencing was used to evaluate mutations in 409 common mutated cancer-related genes in malignant mesothelioma of tunica vaginalis testis (MMTVT) of 81-year-old man. Multifocal papillary-solid areas contained necrosis among highly cellular fields with multiple mitoses. It was positive for WT1, CKAE1/AE3, calretinin, CK7 with negativity for CK5, PSA, TTF-1. Following mutations were revealed in PARP1 (NM_001618: c.2285TG, p.K135R), MTRR (NM_024010: c.147A>G, p.I49M) and two sorts of mutations in structure of KMT2C gene (NM_170606: c.2447_2448insA (c.2447dupA), p.Y816fs and NM_170606: c.1042G>A, p.D348N) for the first time in MMTVT.


Assuntos
Mesotelioma/genética , Neoplasias Testiculares/genética , Adenosina Trifosfatases/genética , Idoso de 80 Anos ou mais , Proteínas de Ligação a DNA/genética , Ferredoxina-NADP Redutase/genética , Humanos , Masculino , Mutação , Fator de Transcrição PAX8/genética , Poli(ADP-Ribose) Polimerase-1/genética , Ubiquitina-Proteína Ligases/genética
8.
J Cancer Res Clin Oncol ; 146(7): 1659-1670, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32279088

RESUMO

PURPOSE: Desmoplastic small round cell tumors (DSRCTs) are highly malignant and very rare soft tissue sarcomas with a high unmet need for new therapeutic options. Therefore, we examined poly(ADP-ribose) polymerase 1 (PARP1) and Schlafen-11 (SLFN11) expression in DSRCT tumor tissue and the combination of PARP inhibitor olaparib with the alkylating agent temozolomide (TMZ) in a preclinical DSRCT model. METHODS: PARP1 and SLFN11 have been described as predictive biomarkers for response to PARP inhibition. Expression of PARP1 and SLFN11 was assessed in 16 and 12 DSRCT tumor tissue samples, respectively. Effects of single-agent olaparib, and olaparib and TMZ combination treatment were examined using the preclinical JN-DSRCT-1 model. In vitro, single-agent and combination treatment effects on cell viability, the cell cycle, DNA damage and apoptosis were examined. Olaparib and TMZ combination treatment was also assessed in vivo. RESULTS: PARP1 and SLFN11 expression was observed in 100% and 92% of DSRCT tumor tissues, respectively. Olaparib treatment reduced cell viability and cell migration in a dose-dependent manner in vitro. Drug synergy between olaparib and TMZ was observed in vitro and in vivo. Combination treatment led to a cell-cycle arrest and induction of DNA damage and apoptosis, even when combined at low dosages. CONCLUSION: We show high PARP1 and SLFN11 expression in DSRCT tumor material and antitumor effects following olaparib and TMZ combination treatment in a preclinical DSRCT model. This suggests that olaparib and TMZ combination treatment could be a potential treatment option for DSRCTs.


Assuntos
Antineoplásicos/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Temozolomida/farmacologia , Adolescente , Adulto , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Criança , Tumor Desmoplásico de Pequenas Células Redondas/tratamento farmacológico , Tumor Desmoplásico de Pequenas Células Redondas/etiologia , Tumor Desmoplásico de Pequenas Células Redondas/metabolismo , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Expressão Gênica , Humanos , Masculino , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
9.
Nat Chem Biol ; 16(7): 791-800, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32251407

RESUMO

Cancer treatment generally involves drugs used in combinations. Most previous work has focused on identifying and understanding synergistic drug-drug interactions; however, understanding antagonistic interactions remains an important and understudied issue. To enrich for antagonism and reveal common features of these combinations, we screened all pairwise combinations of drugs characterized as activators of regulated cell death. This network is strongly enriched for antagonism, particularly a form of antagonism that we call 'single-agent dominance'. Single-agent dominance refers to antagonisms in which a two-drug combination phenocopies one of the two agents. Dominance results from differences in cell death onset time, with dominant drugs acting earlier than their suppressed counterparts. We explored mechanisms by which parthanatotic agents dominate apoptotic agents, finding that dominance in this scenario is caused by mutually exclusive and conflicting use of Poly(ADP-ribose) polymerase 1 (PARP1). Taken together, our study reveals death kinetics as a predictive feature of antagonism, due to inhibitory crosstalk between cell death pathways.


Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Parthanatos/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/genética , Apoptose/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Antagonismo de Drogas , Sinergismo Farmacológico , Humanos , Cinética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Parthanatos/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo
10.
Nat Commun ; 11(1): 752, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029722

RESUMO

Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs of mammalian cells, however, is labor-intensive, time-consuming, and, in some cell types, essentially impossible. Here, we present an approach to create isogenic pairs of cells that avoids single cell cloning, and screen these pairs with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We query the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, identifying both expected and uncharacterized buffering and synthetic lethal interactions. Additionally, we compare acute CRISPR-based knockout, single cell clones, and small-molecule inhibition. We observe that, while the approaches provide largely overlapping information, differences emerge, highlighting an important consideration when employing genetic screens to identify and characterize potential drug targets. We anticipate that this methodology will be broadly useful to comprehensively study gene function across many contexts.


Assuntos
Testes Genéticos/métodos , Apoptose/genética , Sistemas CRISPR-Cas , Linhagem Celular , Células Clonais , Técnicas de Inativação de Genes , Biblioteca Gênica , Redes Reguladoras de Genes , Humanos , Família Multigênica , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/deficiência , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , Análise de Célula Única , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/deficiência , Proteína bcl-X/genética
11.
Arterioscler Thromb Vasc Biol ; 40(4): 914-928, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32078363

RESUMO

OBJECTIVE: Systemic low-grade inflammation associated with obesity and metabolic syndrome is a strong risk factor for the development of diabetes mellitus and associated cardiovascular complications. This inflammatory state is caused by release of proinflammatory cytokines by macrophages, especially in adipose tissue. Long noncoding RNAs regulate macrophage activation and inflammatory gene networks, but their role in macrophage dysfunction during diet-induced obesity has been largely unexplored. Approach and Results: We sequenced total RNA from peritoneal macrophages isolated from mice fed either high-fat diet or standard diet and performed de novo transcriptome assembly to identify novel differentially expressed mRNAs and long noncoding RNAs. A top candidate long noncoding RNA, macrophage inflammation-suppressing transcript (Mist), was downregulated in both peritoneal macrophages and adipose tissue macrophages from high-fat diet-fed mice. GapmeR-mediated Mist knockdown in vitro and in vivo upregulated expression of genes associated with immune response and inflammation and increased modified LDL (low-density lipoprotein) uptake in macrophages. Conversely, Mist overexpression decreased basal and LPS (lipopolysaccharide)-induced expression of inflammatory response genes and decreased modified LDL uptake. RNA-pull down coupled with mass spectrometry showed that Mist interacts with PARP1 (poly [ADP]-ribose polymerase-1). Disruption of this RNA-protein interaction increased PARP1 recruitment and chromatin PARylation at promoters of inflammatory genes, resulting in increased gene expression. Furthermore, human orthologous MIST was also downregulated by proinflammatory stimuli, and its expression in human adipose tissue macrophages inversely correlated with obesity and insulin resistance. CONCLUSIONS: Mist is a novel protective long noncoding RNA, and its loss during obesity contributes to metabolic dysfunction and proinflammatory phenotype of macrophages via epigenetic mechanisms.


Assuntos
Inflamação/fisiopatologia , Ativação de Macrófagos/genética , Obesidade/genética , Obesidade/fisiopatologia , RNA Longo não Codificante/fisiologia , Tecido Adiposo/metabolismo , Animais , Linhagem Celular , LDL-Colesterol/metabolismo , Cromatina/genética , Citocinas/fisiologia , Regulação para Baixo , Humanos , Metabolismo dos Lipídeos/genética , Masculino , Síndrome Metabólica/genética , Síndrome Metabólica/fisiopatologia , Camundongos Endogâmicos C57BL , Poli(ADP-Ribose) Polimerase-1/genética , Poli ADP Ribosilação , Regulação para Cima
12.
Oxid Med Cell Longev ; 2020: 5367102, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32104534

RESUMO

Treatments on neoplastic diseases and cancer using genotoxic drugs often cause long-term health problems related to premature aging. The underlying mechanism is poorly understood. Based on the study of a long-lasting senescence-like growth arrest (10-12 weeks) of human dermal fibroblasts induced by psoralen plus UVA (PUVA) treatment, we here revealed that slowly repaired bulky DNA damages can serve as a "molecular scar" leading to reduced cell proliferation through persistent endogenous production of reactive oxygen species (ROS) that caused accelerated telomere erosion. The elevated levels of ROS were the results of mitochondrial dysfunction and the activation of NADPH oxidase (NOX). A combined inhibition of DNA-PK and PARP1 could suppress the level of ROS. Together with a reduced expression level of BRCA1 as well as the upregulation of PP2A and 53BP1, these data suggest that the NHEJ repair of DNA double-strand breaks may be the initial trigger of metabolic changes leading to ROS production. Further study showed that stimulation of the pentose phosphate pathway played an important role for NOX activation, and ROS could be efficiently suppressed by modulating the NADP/NADPH ratio. Interestingly, feeding cells with ribose-5-phosphate, a precursor for nucleotide biosynthesis that produced through the PPP, could evidently suppress the ROS level and prevent the cell enlargement related to mitochondrial biogenesis. Taken together, these results revealed an important signaling pathway between DNA damage repair and the cell metabolism, which contributed to the premature aging effects of PUVA, and may be generally applicable for a large category of chemotherapeutic reagents including many cancer drugs.


Assuntos
Senescência Celular/fisiologia , Dano ao DNA/fisiologia , Estresse Oxidativo/fisiologia , Células Cultivadas , Senescência Celular/genética , Dano ao DNA/genética , Reparo do DNA/genética , Reparo do DNA/fisiologia , Humanos , NADP/genética , NADP/metabolismo , Oxirredução , Estresse Oxidativo/genética , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ribosemonofosfatos/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
13.
Sci Rep ; 10(1): 2585, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066817

RESUMO

Polypharmacology plays an important role in defining response and adverse effects of drugs. For some mechanisms, experimentally mapping polypharmacology is commonplace, although this is typically done within the same protein class. Four PARP inhibitors have been approved by the FDA as cancer therapeutics, yet a precise mechanistic rationale to guide clinicians on which to choose for a particular patient is lacking. The four drugs have largely similar PARP family inhibition profiles, but several differences at the molecular and clinical level have been reported that remain poorly understood. Here, we report the first comprehensive characterization of the off-target kinase landscape of four FDA-approved PARP drugs. We demonstrate that all four PARP inhibitors have a unique polypharmacological profile across the kinome. Niraparib and rucaparib inhibit DYRK1s, CDK16 and PIM3 at clinically achievable, submicromolar concentrations. These kinases represent the most potently inhibited off-targets of PARP inhibitors identified to date and should be investigated further to clarify their potential implications for efficacy and safety in the clinic. Moreover, broad kinome profiling is recommended for the development of PARP inhibitors as PARP-kinase polypharmacology could potentially be exploited to modulate efficacy and side-effect profiles.


Assuntos
Antineoplásicos/química , Indazóis/química , Indóis/química , Ftalazinas/química , Piperazinas/química , Piperidinas/química , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/química , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Sítios de Ligação , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Células HEK293 , Humanos , Indazóis/administração & dosagem , Indazóis/efeitos adversos , Indóis/administração & dosagem , Indóis/efeitos adversos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Ftalazinas/administração & dosagem , Ftalazinas/efeitos adversos , Piperazinas/administração & dosagem , Piperazinas/efeitos adversos , Piperidinas/administração & dosagem , Piperidinas/efeitos adversos , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Inibidores de Poli(ADP-Ribose) Polimerases/efeitos adversos , Polifarmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Especificidade por Substrato
14.
Sci Rep ; 10(1): 3009, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080212

RESUMO

Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 3 (HER3) have been investigated as triple-negative breast cancer (TNBC) biomarkers. Reduced EGFR levels can be compensated by increases in HER3; thus, assaying EGFR and HER3 together may improve prognostic value. In a multi-institutional cohort of 510 TNBC patients, we analyzed the impact of HER3, EGFR, or combined HER3-EGFR protein expression in pre-treatment samples on breast cancer-specific and distant metastasis-free survival (BCSS and DMFS, respectively). A subset of 60 TNBC samples were RNA-sequenced using massive parallel sequencing. The combined HER3-EGFR score outperformed individual HER3 and EGFR scores, with high HER3-EGFR score independently predicting worse BCSS (Hazard Ratio [HR] = 2.30, p = 0.006) and DMFS (HR = 1.78, p = 0.041, respectively). TNBCs with high HER3-EGFR scores exhibited significantly suppressed ATM signaling and differential expression of a network predicted to be controlled by low TXN activity, resulting in activation of EGFR, PARP1, and caspases and inhibition of p53 and NFκB. Nuclear PARP1 protein levels were higher in HER3-EGFR-high TNBCs based on immunohistochemistry (p = 0.036). Assessing HER3 and EGFR protein expression in combination may identify which adjuvant chemotherapy-treated TNBC patients have a higher risk of treatment resistance and may benefit from a dual HER3-EGFR inhibitor and a PARP1 inhibitor.


Assuntos
Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Receptor ErbB-3/genética , Neoplasias de Mama Triplo Negativas/genética , Adulto , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Estudos de Coortes , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Pessoa de Meia-Idade , NF-kappa B/genética , NF-kappa B/metabolismo , Estadiamento de Neoplasias , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Prognóstico , Receptor ErbB-3/metabolismo , Análise de Sobrevida , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Neoplasias de Mama Triplo Negativas/diagnóstico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/mortalidade , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
15.
BMB Rep ; 53(2): 112-117, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31964471

RESUMO

A recent study suggested that methylation of ubiquitin-like with PHD and RING finger domain 1 (UHRF1) is regulated by SET7 and lysine-specific histone demethylase 1A (LSD1) and is essential for homologous recombination (HR). The study demonstrated that SET7-mediated methylation of UHRF1 promotes polyubiquitination of proliferating cell nuclear antigen (PCNA), inducing HR. However, studies on mediators that interact with and recruit UHRF1 to damaged lesions are needed to elucidate the mechanism of UHRF1 methylationinduced HR. Here, we identified that poly [ADP-ribose] polymerase 1 (PARP1) interacts with damage-induced methylated UHRF1 specifically and mediates UHRF1 to induce HR progression. Furthermore, cooperation of UHRF1-PARP1 is essential for cell viability, suggesting the importance of the interaction of UHRF1-PARP1 for damage tolerance in response to damage. Our data revealed that PARP1 mediates the HR mechanism, which is regulated by UHRF1 methylation. The data also indicated the significant role of PARP1 as a mediator of UHRF1 methylation-correlated HR pathway. [BMB Reports 2020; 53(2): 112-117].


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Dano ao DNA/genética , Recombinação Homóloga/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/genética , Sobrevivência Celular/genética , Dano ao DNA/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Células HCT116 , Células HEK293 , Humanos , Peróxido de Hidrogênio/farmacologia , Poli(ADP-Ribose) Polimerase-1/genética , Ligação Proteica , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética
16.
Proc Natl Acad Sci U S A ; 117(4): 2032-2042, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31932422

RESUMO

Resistance to androgen deprivation therapy, or castration-resistant prostate cancer (CRPC), is often accompanied by metastasis and is currently the ultimate cause of prostate cancer-associated deaths in men. Recently, secondary hormonal therapies have led to an increase of neuroendocrine prostate cancer (NEPC), a highly aggressive variant of CRPC. Here, we identify that high levels of cell surface receptor Trop2 are predictive of recurrence of localized prostate cancer. Moreover, Trop2 is significantly elevated in CRPC and NEPC, drives prostate cancer growth, and induces neuroendocrine phenotype. Overexpression of Trop2 induces tumor growth and metastasis while loss of Trop2 suppresses these abilities in vivo. Trop2-driven NEPC displays a significant up-regulation of PARP1, and PARP inhibitors significantly delay tumor growth and metastatic colonization and reverse neuroendocrine features in Trop2-driven NEPC. Our findings establish Trop2 as a driver and therapeutic target for metastatic prostate cancer with neuroendocrine phenotype and suggest that high Trop2 levels could identify cancers that are sensitive to Trop2-targeting therapies and PARP1 inhibition.


Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/secundário , Carcinoma Neuroendócrino/patologia , Moléculas de Adesão Celular/metabolismo , Regulação Neoplásica da Expressão Gênica , Poli(ADP-Ribose) Polimerase-1/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Animais , Antígenos de Neoplasias/genética , Apoptose , Biomarcadores Tumorais/genética , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Carcinoma Neuroendócrino/tratamento farmacológico , Carcinoma Neuroendócrino/metabolismo , Moléculas de Adesão Celular/genética , Movimento Celular , Proliferação de Células , Seguimentos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Fenótipo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Prognóstico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Asian Pac J Cancer Prev ; 21(1): 43-48, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31983162

RESUMO

OBJECTIVES: To evaluate the association of allelic and genotypic frequencies of PSCA (rs2976392), TNF-α (rs1800629), PARP1 (rs1136410) and TP53 (rs368771578) SNPs with GC susceptibility in a Brazilian population. MATERIALS AND METHODS: This is a retrospective study, which included 102 paraffin-embedded adenocarcinoma tissue samples > 5 years of obtention, with 204 alleles for each studied SNP. Other 102 healthy tissue samples were included as controls. For analysis, the genotyping method Dideoxy Single Allele-Specific - PCR was used. Statistical analysis was performed with the Bioestat software 5.3, determining Hardy-Weinberg's equilibrium for the genotypic frequencies p-values < 0.05 were considered significant. RESULTS: PSCA (rs2976392) and TNF-α (rs1800629) SNPs were associated with GC in the analyzed samples (X2=10.3/102 and p<0.001/0.00001, respectively). TNF-α (rs1800629) SNP presented also a statistically significant relationship between its genotypes and the morphological pattern (intestinal/diffuse) (p<0.032). However, PARP1 (rs1136410) and TP53 (rs368771578) SNPs were in Hardy-Weinberg's equilibrium and, therefore, were not significantly associated with GC in these samples (X2=0.73/2.89 and p<0.39/0.08). CONCLUSIONS: PSCA (rs2976392) and TNF-α (rs1800629) SNPs are potential molecular markers of susceptibility to GC development. PARP1 (rs1136410) and TP53 (rs368771578) SNPs were not associated with the risk of GC development.


Assuntos
Antígenos de Neoplasias/genética , Predisposição Genética para Doença/genética , Proteínas de Neoplasias/genética , Poli(ADP-Ribose) Polimerase-1/genética , Polimorfismo de Nucleotídeo Único/genética , Neoplasias Gástricas/genética , Fator de Necrose Tumoral alfa/genética , Proteína Supressora de Tumor p53/genética , Adenocarcinoma/genética , Alelos , Biomarcadores Tumorais/genética , Brasil , Estudos de Casos e Controles , Feminino , Proteínas Ligadas por GPI/genética , Frequência do Gene/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
18.
Cell Death Dis ; 11(1): 71, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992690

RESUMO

Poly(ADP-ribose) polymerase 1 (PARP1) regulates gene transcription in addition to functioning as a DNA repair factor. Forkhead box O1 (FoxO1) is a transcription factor involved in extensive biological processes. Here, we report that PARP1 binds to two separate motifs on the FoxO1 promoter and represses its transcription in a polymerase-independent manner. Using PARP1-knock out (KO) cells, wild-type-PARP1-complemented cells and catalytic mutant PARP1E988K-reconstituted cells, we investigated transcriptional regulation by PARP1. PARP1 loss led to reduced DNA damage response and ~362-fold resistance to five PARP inhibitors (PARPis) in Ewing sarcoma cells. RNA sequencing showed 492 differentially expressed genes in a PARP1-KO subline, in which the FoxO1 mRNA levels increased up to more than five times. The change in the FoxO1 expression was confirmed at both mRNA and protein levels in different PARP1-KO and complemented cells. Moreover, exogenous PARP1 overexpression reduced the endogenous FoxO1 protein in RD-ES cells. Competitive EMSA and ChIP assays revealed that PARP1 specifically bound to the FoxO1 promoter. DNase I footprinting, mutation analyses, and DNA pulldown FREP assays showed that PARP1 bound to two particular nucleotide sequences separately located at -813 to -826 bp and -1805 to -1828 bp regions on the FoxO1 promoter. Either the PARPi olaparib or the PARP1 catalytic mutation (E988K) did not impair the repression of PARP1 on the FoxO1 expression. Exogenous FoxO1 overexpression did not impair cellular PARPi sensitivity. These findings demonstrate a new PARP1-gene promoter binding mode and a new transcriptional FoxO1 gene repressor.


Assuntos
Proteína Forkhead Box O1/genética , Regulação da Expressão Gênica/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Sarcoma de Ewing/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Dano ao DNA/efeitos dos fármacos , Proteína Forkhead Box O1/metabolismo , Técnicas de Inativação de Genes , Humanos , Mutação , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , Sarcoma de Ewing/genética , Regulação para Cima
19.
Cell Death Dis ; 11(1): 22, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924749

RESUMO

Accelerated atherosclerotic calcification is responsible for plaque burden, especially in diabetes. The regulatory mechanism for atherosclerotic calcification in diabetes is poorly characterized. Here we show that deletion of PARP-1, a main enzyme in diverse metabolic complications, attenuates diabetic atherosclerotic calcification and decreases vessel stiffening in mice through Runx2 suppression. Specifically, PARP-1 deficiency reduces diabetic arteriosclerotic calcification by regulating Stat1-mediated synthetic phenotype switching of vascular smooth muscle cells and macrophage polarization. Meanwhile, both vascular smooth muscle cells and macrophages manifested osteogenic differentiation in osteogenic media, which was attenuated by PARP-1/Stat1 inhibition. Notably, Stat1 acts as a positive transcription factor by directly binding to the promoter of Runx2 and promoting atherosclerotic calcification in diabetes. Our results identify a new function of PARP-1, in which metabolism disturbance-related stimuli activate the Runx2 expression mediated by Stat1 transcription to facilitate diabetic arteriosclerotic calcification. PARP-1 inhibition may therefore represent a useful therapy for this challenging complication.


Assuntos
Aterosclerose/enzimologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Complicações do Diabetes/enzimologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Fator de Transcrição STAT1/metabolismo , Calcificação Vascular/enzimologia , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Complicações do Diabetes/genética , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Osteogênese/genética , Fenantrenos/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , Fator de Transcrição STAT1/genética , Calcificação Vascular/genética
20.
Ann Diagn Pathol ; 44: 151442, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31855806

RESUMO

Desmoid-type fibromatoses (or desmoid tumors) are entities of intermediate biological potential and are locally invasive. Radical surgery, as state of the art therapy, is frequently limited by incomplete resections. Hormone modifying therapies are promising but further research is required. Poly Adenosine Diphosphate Ribose Polymerase-1 (PARP-1), a DNA repairing enzyme, might be a pathogenetic factor and could become a potential target for therapy as shown by the successful treatment of selected carcinomas and sarcomas by PARP-inhibition. In this study, we investigated the expression of estrogen receptors (ER) α (1) and ß (2), progesterone receptor (PR), androgen receptor (AR), as well as PARP-1 via immunohistochemistry and quantitative RT-PCR in 69 tissue samples of desmoid tumors. Immunohistochemistry was quantified using the Immunoreactivity Score (IRS). Overall expression patterns were correlated with clinical-pathologic parameters to determine their value as a prognostic factor. Among the investigated hormone receptors only ERß showed partial cytoplasmic reactivity. PARP-1 revealed variable nuclear positivity with IRS ranging from 0 to 6. Univariate survival analysis showed that higher expression of estrogen receptor 1 was associated with shorter disease-free survival (p = 0.005). Uni- (p = 0.03) and multivariate (p = 0.003) analyses of mRNA data revealed that higher PARP-1 expression correlated with earlier recurrence. According to this study PARP-1 expression is associated with poorer prognosis, that is faster recurrence, highlighting the possibility of PARP-1-targeting agents as a therapeutic option. Hormone receptors were of minor prognostic relevance in this study.


Assuntos
Biomarcadores Tumorais/metabolismo , Fibromatose Agressiva/diagnóstico , Poli(ADP-Ribose) Polimerase-1/metabolismo , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Intervalo Livre de Doença , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Fibromatose Agressiva/metabolismo , Fibromatose Agressiva/patologia , Humanos , Imuno-Histoquímica , Lactente , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , Prognóstico , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Adulto Jovem
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