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1.
Nature ; 571(7766): 521-527, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270457

RESUMO

The integrity of genomes is constantly threatened by problems encountered by the replication fork. BRCA1, BRCA2 and a subset of Fanconi anaemia proteins protect stalled replication forks from degradation by nucleases, through pathways that involve RAD51. The contribution and regulation of BRCA1 in replication fork protection, and how this role relates to its role in homologous recombination, is unclear. Here we show that BRCA1 in complex with BARD1, and not the canonical BRCA1-PALB2 interaction, is required for fork protection. BRCA1-BARD1 is regulated by a conformational change mediated by the phosphorylation-directed prolyl isomerase PIN1. PIN1 activity enhances BRCA1-BARD1 interaction with RAD51, thereby increasing the presence of RAD51 at stalled replication structures. We identify genetic variants of BRCA1-BARD1 in patients with cancer that exhibit poor protection of nascent strands but retain homologous recombination proficiency, thus defining domains of BRCA1-BARD1 that are required for fork protection and associated with cancer development. Together, these findings reveal a BRCA1-mediated pathway that governs replication fork protection.


Assuntos
Proteína BRCA1/química , Proteína BRCA1/metabolismo , Replicação do DNA , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína BRCA1/genética , Linhagem Celular Tumoral , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Isomerismo , Mutação , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Rad51 Recombinase/metabolismo
2.
Int J Mol Med ; 43(6): 2491-2498, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31017265

RESUMO

Nucleos(t)ide analogues (NAs) are currently the most important anti­viral treatment option for patients with chronic hepatitis B (CHB). Adefovir dipivoxil (ADV), a diester pro­drug of adefovir, has been widely used for the clinical therapy of hepatitis B virus infection. It has been previously reported that adefovir induced chromosomal aberrations (CAs) in the in vitro human peripheral blood lymphocyte assay, while the genotoxic mechanism remains elusive. To evaluate the possible mechanisms, the genotoxic effects of ADV on the TK6 and DT40 cell lines, as well as DNA repair­deficient variants of DT40 cells, were assessed in the present study. A karyotype assay revealed ADV­induced CAs, particularly chromosomal breaks, in wild­type DT40 and TK6 cells. A γ­H2AX foci formation assay confirmed the presence of DNA damage following treatment with ADV. Furthermore, Brca1­/­ DT40 cells exhibited an increased sensitivity to ADV, while the knockdown of various other DNA damage­associated genes did not markedly affect the sensitivity. These comprehensive genetic studies identified the genotoxic capacity of ADV and suggested that Brca1 may be involved in the tolerance of ADV­induced DNA damage. These results may contribute to the development of novel drugs against CHB with higher therapeutic efficacy and less genotoxicity.


Assuntos
Adenina/análogos & derivados , Antivirais/efeitos adversos , Proteína BRCA1/metabolismo , Aberrações Cromossômicas/induzido quimicamente , Dano ao DNA/efeitos dos fármacos , Organofosfonatos/efeitos adversos , Adenina/efeitos adversos , Proteína BRCA1/genética , Linhagem Celular , Deleção de Genes , Hepatite B Crônica/tratamento farmacológico , Humanos
3.
Artif Cells Nanomed Biotechnol ; 47(1): 1101-1112, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30942098

RESUMO

Cases of more than three primary cancers are very rare. This study analyzed the genetic susceptibility of gene polymorphisms in three patients with multiple primary malignant neoplasms and examined the possible pathogenesis. The clinical data and whole genome sequence of three patients (1 with 5 primary cancers, 1 with 4 primary cancers, and 1 with 3 primary cancers) were aligned with a series of databases. We found the three patients contained a total of seven types of malignant tumours (endometrial cancer, ovarian cancer, breast cancer, colon cancer, ureter cancer, bladder cancer and kidney cancer). It was found that the varied genes in Patient 1 (5 primary cancers) were BRIP1, FANCG, NBN, AXIN2, SRD5A2, and CEBPA. Patient 2 (4 primary cancers) had variations in the following genes: BMPR1A, FANCD2, MLH3, BRCA2, and FANCM. Patient 3 (3 primary cancers) had variations in the following genes: MEN1, ATM, MSH3, BRCA1, FANCL, CEBPA, and FANCA. String software was used to analyze the KEGG pathway of the variations in these three samples, which revealed that the genes are involved in the Fanconi anaemia pathway. Defects in DNA damage repair may be one of the causes of multiple primary cancers.


Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Neoplasias Primárias Múltiplas/genética , Polimorfismo Genético , Idoso , Análise Mutacional de DNA , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Primárias Múltiplas/metabolismo , Software
4.
J Cancer Res Clin Oncol ; 145(6): 1471-1484, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31020420

RESUMO

PURPOSE: To study an association between IL-6 signaling and resistance to radiotherapy of prostate cancer (PCa) and explore the molecular mechanisms involved. METHODS: IL-6 expressing C4-2 and CWR22Rv1 (C4-2IL-6/CWRIL-6) and vector control (C4-2vec/CWRvec) cell lines were developed. Radiation-sensitivities of these cells were compared in clonogenic assay, Comet assay, and γH2AX staining. In xenograft animal studies, radiation-sensitivity of C4-2IL-6 cell-derived tumors vs. C4-2vec cell-derived tumors was investigated. To reveal IL-6 downstream molecules involved in DNA repair after radiation, qPCR and Western blot analyses as well as immunofluorescence staining were performed. Transcriptional control of IL-6 on ATM and ATR molecules was also investigated. RESULTS: We found C4-2IL-6 and CWRIL-6 cells survived better than their vector control cells after irradiation, and animal studies confirmed such in vitro results. We discovered that DNA repair-related molecules such as ATM, ATR, BRCA1, and BRCA2 were significantly upregulated in irradiated IL-6 expressing cells compared with vector control cells. We further defined that IL-6 signaling regulated cellular expressions of ATM and ATR at the transcriptional level through the activation of Stat3 signaling pathway. CONCLUSIONS: IL-6 leads to PCa resistance to radiation through upregulation of DNA repair associated molecules ATM, ATR, BRCA1, and BRCA2.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Reparo do DNA , Interleucina-6/metabolismo , Neoplasias da Próstata/radioterapia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Nus , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Tolerância a Radiação , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
5.
PLoS Genet ; 15(3): e1008049, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30925164

RESUMO

The BARD1 protein, which heterodimerizes with BRCA1, is encoded by a known breast cancer susceptibility gene. While several BARD1 variants have been identified as pathogenic, many more missense variants exist that do not occur frequently enough to assign a clinical risk. In this paper, whole exome sequencing of over 10,000 cancer samples from 33 cancer types identified from somatic mutations and loss of heterozygosity in tumors 76 potentially cancer-associated BARD1 missense and truncation variants. These variants were tested in a functional assay for homology-directed repair (HDR), as HDR deficiencies have been shown to correlate with clinical pathogenicity for BRCA1 variants. From these 76 variants, 4 in the ankyrin repeat domain and 5 in the BRCT domain were found to be non-functional in HDR. Two known benign variants were found to be functional in HDR, and three known pathogenic variants were non-functional, supporting the notion that the HDR assay can be used to predict the clinical risk of BARD1 variants. The identification of HDR-deficient variants in the ankyrin repeat domain indicates there are DNA repair functions associated with this domain that have not been closely examined. In order to examine whether BARD1-associated loss of HDR function results in DNA damage sensitivity, cells expressing non-functional BARD1 variants were treated with ionizing radiation or cisplatin. These cells were found to be more sensitive to DNA damage, and variations in the residual HDR function of non-functional variants did not correlate with variations in sensitivity. These findings improve the understanding of BARD1 functional domains in DNA repair and support that this functional assay is useful for predicting the cancer association of BARD1 variants.


Assuntos
Neoplasias/genética , Reparo de DNA por Recombinação/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Animais , Proteína BRCA1/metabolismo , Gatos , Dano ao DNA , Reparo do DNA/genética , Cães , Feminino , Humanos , Camundongos , Mutação de Sentido Incorreto/genética , Alinhamento de Sequência , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Sequenciamento Completo do Exoma
6.
Nature ; 567(7749): 545-549, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30894746

RESUMO

MYC is an oncogenic transcription factor that binds globally to active promoters and promotes transcriptional elongation by RNA polymerase II (RNAPII)1,2. Deregulated expression of the paralogous protein MYCN drives the development of neuronal and neuroendocrine tumours and is often associated with a particularly poor prognosis3. Here we show that, similar to MYC, activation of MYCN in human neuroblastoma cells induces escape of RNAPII from promoters. If the release of RNAPII from transcriptional pause sites (pause release) fails, MYCN recruits BRCA1 to promoter-proximal regions. Recruitment of BRCA1 prevents MYCN-dependent accumulation of stalled RNAPII and enhances transcriptional activation by MYCN. Mechanistically, BRCA1 stabilizes mRNA decapping complexes and enables MYCN to suppress R-loop formation in promoter-proximal regions. Recruitment of BRCA1 requires the ubiquitin-specific protease USP11, which binds specifically to MYCN when MYCN is dephosphorylated at Thr58. USP11, BRCA1 and MYCN stabilize each other on chromatin, preventing proteasomal turnover of MYCN. Because BRCA1 is highly expressed in neuronal progenitor cells during early development4 and MYC is less efficient than MYCN in recruiting BRCA1, our findings indicate that a cell-lineage-specific stress response enables MYCN-driven tumours to cope with deregulated RNAPII function.


Assuntos
Proteína BRCA1/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , Estabilidade Proteica , Tioléster Hidrolases/metabolismo
7.
Proc Natl Acad Sci U S A ; 116(15): 7363-7370, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30918126

RESUMO

Coordination of growth and genomic stability is critical for normal cell physiology. Although the E3 ubiquitin ligase BRCA1 is a key player in maintenance of genomic stability, its role in growth signaling remains elusive. Here, we show that BRCA1 facilitates stabilization of YAP1 protein and turning "off" the Hippo pathway through ubiquitination of NF2. In BRCA1-deficient cells Hippo pathway is "turned On." Phosphorylation of YAP1 is crucial for this signaling process because a YAP1 mutant harboring alanine substitutions (Mt-YAP5SA) in LATS1 kinase recognition sites not only resists degradation but also rescues YAP1 transcriptional activity in BRCA1-deficient cells. Furthermore, an ectopic expression of the active Mt-YAP5SA, but not inactive Mt-YAP6SA, promotes EGF-independent proliferation and tumorigenesis in BRCA1-/- mammary epithelial cells. These findings establish an important role of BRCA1 in regulating stability of YAP1 protein that correlates positively with cell proliferation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína BRCA1/metabolismo , Neoplasias da Mama/metabolismo , Neurofibromina 2/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Adaptadoras de Transdução de Sinal/genética , Substituição de Aminoácidos , Proteína BRCA1/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Neurofibromina 2/genética , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética
8.
Nat Cell Biol ; 21(3): 311-318, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30804502

RESUMO

Genotoxic DNA double-strand breaks (DSBs) can be repaired by error-free homologous recombination (HR) or mutagenic non-homologous end-joining1. HR supresses tumorigenesis1, but is restricted to the S and G2 phases of the cell cycle when a sister chromatid is present2. Breast cancer type 1 susceptibility protein (BRCA1) promotes HR by antagonizing the anti-resection factor TP53-binding protein 1(53BP1) (refs. 2-5), but it remains unknown how BRCA1 function is limited to the S and G2 phases. We show that BRCA1 recruitment requires recognition of histone H4 unmethylated at lysine 20 (H4K20me0), linking DSB repair pathway choice directly to sister chromatid availability. We identify the ankyrin repeat domain of BRCA1-associated RING domain protein 1 (BARD1)-the obligate BRCA1 binding partner3-as a reader of H4K20me0 present on new histones in post-replicative chromatin6. BARD1 ankyrin repeat domain mutations disabling H4K20me0 recognition abrogate accumulation of BRCA1 at DSBs, causing aberrant build-up of 53BP1, and allowing anti-resection activity to prevail in S and G2. Consequently, BARD1 recognition of H4K20me0 is required for HR and resistance to poly (ADP-ribose) polymerase inhibitors. Collectively, this reveals that BRCA1-BARD1 monitors the replicative state of the genome to oppose 53BP1 function, routing only DSBs within sister chromatids to HR.


Assuntos
Proteína BRCA1/metabolismo , Cromátides/metabolismo , Histonas/metabolismo , Recombinação Homóloga , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Proteína BRCA1/genética , Linhagem Celular Tumoral , Cromátides/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Fase G2/genética , Células HCT116 , Células HeLa , Humanos , Lisina/metabolismo , Metilação , Fase S/genética , Homologia de Sequência de Aminoácidos , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética
9.
J Biol Chem ; 294(15): 5980-5992, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30765603

RESUMO

Genetic testing for BRCA1, a DNA repair protein, can identify carriers of pathogenic variants associated with a substantially increased risk for breast and ovarian cancers. However, an association with increased risk is unclear for a large fraction of BRCA1 variants present in the human population. Most of these variants of uncertain clinical significance lead to amino acid changes in the BRCA1 protein. Functional assays are valuable tools to assess the potential pathogenicity of these variants. Here, we systematically probed the effects of substitutions in the C terminus of BRCA1: the N- and C-terminal borders of its tandem BRCT domain, the BRCT-[N-C] linker region, and the α1 and α'1 helices in BRCT-[N] and -[C]. Using a validated transcriptional assay based on a fusion of the GAL4 DNA-binding domain to the BRCA1 C terminus (amino acids 1396-1863), we assessed the functional impact of 99 missense variants of BRCA1. We include the data obtained for these 99 missense variants in a joint analysis to generate the likelihood of pathogenicity for 347 missense variants in BRCA1 using VarCall, a Bayesian integrative statistical model. The results from this analysis increase our understanding of BRCA1 regions less tolerant to changes, identify functional borders of structural domains, and predict the likelihood of pathogenicity for 98% of all BRCA1 missense variants in this region recorded in the population. This knowledge will be critical for improving risk assessment and clinical treatment of carriers of BRCA1 variants.


Assuntos
Proteína BRCA1 , Neoplasias da Mama , Modelos Moleculares , Mutação de Sentido Incorreto , Neoplasias Ovarianas , Substituição de Aminoácidos , Proteína BRCA1/química , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Células HEK293 , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Domínios Proteicos , Relação Estrutura-Atividade
10.
Genes Dev ; 33(7-8): 436-451, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30804228

RESUMO

Caenorhabditis elegans has two histone H3 Lys9 methyltransferases, MET-2 (SETDB1 homolog) and SET-25 (G9a/SUV39H1 related). In worms, we found simple repeat sequences primarily marked by H3K9me2, while transposable elements and silent tissue-specific genes bear H3K9me3. RNA sequencing (RNA-seq) in histone methyltransferase (HMT) mutants shows that MET-2-mediated H3K9me2 is necessary for satellite repeat repression, while SET-25 silences a subset of transposable elements and tissue-specific genes through H3K9me3. A genome-wide synthetic lethality screen showed that RNA processing, nuclear RNA degradation, the BRCA1/BARD1 complex, and factors mediating replication stress survival are necessary for germline viability in worms lacking MET-2 but not SET-25. Unlike set-25 mutants, met-2-null worms accumulated satellite repeat transcripts, which form RNA:DNA hybrids on repetitive sequences, additively with the loss of BRCA1 or BARD1. BRCA1/BARD1-mediated H2A ubiquitination and MET-2 deposited H3K9me2 on satellite repeats are partially interdependent, suggesting both that the loss of silencing generates BRCA-recruiting DNA damage and that BRCA1 recruitment by damage helps silence repeats. The artificial induction of MSAT1 transcripts can itself trigger damage-induced germline lethality in a wild-type background, arguing that the synthetic sterility upon BRCA1/BARD1 and H3K9me2 loss is directly linked to the DNA damage provoked by unscheduled satellite repeat transcription.


Assuntos
Proteína BRCA1/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Histonas/genética , Animais , Proteína BRCA1/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Elementos de DNA Transponíveis/genética , Embrião não Mamífero , Fertilidade/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Repetições de Microssatélites/genética , Mutação , Processamento Pós-Transcricional do RNA/genética , Temperatura Ambiente
11.
Breast Cancer Res Treat ; 175(1): 117-127, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30671767

RESUMO

PURPOSE: We aimed to explore the expression of DNA damage response machinery proteins and their integrated prognostic value in different subgroups of breast cancer. METHODS: Expression of NBS1, BRCA1, BRCA2, ATM, and p53 was determined by immunohistochemistry in 419 surgically resected breast tumors. RESULTS: Loss of NBS1, BRCA1, ATM, and abnormal p53 expression was significantly associated with lower disease-free survival rates. Abnormal DNA damage response protein expression, defined as loss of any one of NBS1, BRCA1, ATM, and/or abnormal p53 expression, was observed in 258 of 399 evaluable cases (64.7%) and was significantly associated with higher tumor grade, larger tumor size, and ER-negative, and/or PR-negative status. Most patients with luminal B (86.1%), HER2-enriched (94.4%), and triple-negative (86.8%) breast cancers had abnormal DNA damage response protein expression. In contrast, abnormal DNA damage response protein expression was found in only 53.8% of luminal A tumors. Abnormal DNA damage response protein expression was associated with significantly lower 5-year disease-free survival rates in all patients (95.6% vs. 84.8%, p = 0.001), as well as in the luminal A subgroup (97.4% vs. 89.0%, p = 0.011). In multivariate analysis, abnormal DNA damage response protein expression remained an independent predictor of shorter disease-free survival for luminal A subtype (hazard ratio 3.14, 95% confidence interval 1.16-8.47; p = 0.024). CONCLUSION: Abnormal DNA damage response protein expression is found in most luminal B and HER2-enriched breast cancers as frequently as in triple-negative breast cancer. In the luminal A subtype, abnormal DNA damage response protein expression is an independent prognostic marker.


Assuntos
Biomarcadores Tumorais , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Dano ao DNA , Adulto , Idoso , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Reparo do DNA , Feminino , Recombinação Homóloga , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico
12.
Mol Cell ; 73(5): 885-899.e6, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30686591

RESUMO

BRCA1 or BRCA2 inactivation drives breast and ovarian cancer but also creates vulnerability to poly(ADP-ribose) polymerase (PARP) inhibitors. To search for additional targets whose inhibition is synthetically lethal in BRCA2-deficient backgrounds, we screened two pairs of BRCA2 isogenic cell lines with DNA-repair-focused small hairpin RNA (shRNA) and CRISPR (clustered regularly interspaced short palindromic repeats)-based libraries. We found that BRCA2-deficient cells are selectively dependent on multiple pathways including base excision repair, ATR signaling, and splicing. We identified APEX2 and FEN1 as synthetic lethal genes with both BRCA1 and BRCA2 loss of function. BRCA2-deficient cells require the apurinic endonuclease activity and the PCNA-binding domain of Ape2 (APEX2), but not Ape1 (APEX1). Furthermore, BRCA2-deficient cells require the 5' flap endonuclease but not the 5'-3' exonuclease activity of Fen1, and chemically inhibiting Fen1 selectively targets BRCA-deficient cells. Finally, we developed a microhomology-mediated end-joining (MMEJ) reporter and showed that Fen1 participates in MMEJ, underscoring the importance of MMEJ as a collateral repair pathway in the context of homologous recombination (HR) deficiency.


Assuntos
Proteína BRCA2/genética , Sistemas CRISPR-Cas , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases Flap/genética , Genes Letais , Neoplasias/genética , Interferência de RNA , Mutações Sintéticas Letais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Morte Celular , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA por Junção de Extremidades , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases Flap/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/genética
13.
BMC Cancer ; 19(1): 44, 2019 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-30630446

RESUMO

BACKGROUND: Poly (ADP-ribose) polymerase inhibitors (PARPi) have become the first targeted therapies available in the treatment of patients with high-grade serous ovarian cancer (HGSOC). We recently described a significant reduction in PARP1 protein levels in vitro and in vivo in patients treated with standard carboplatinum-paclitaxel chemotherapy, raising the question whether the sequence of treatment used today with chemotherapy followed by PARPi is optimal. In this study, we aim to evaluate if the sequence of PARPi followed by chemotherapy could be more beneficial. METHODS: BRCA1-mutated (UWB1.287, SNU-251), epigenetically-silenced (OVCAR8), and wild-type (SKOV3, A2780PAR & A2780CR) ovarian cancer cell lines were exposed to clinically relevant doses of PARPi followed by different doses of standard chemotherapy and compared to the inverse treatment. The therapeutic efficacy was assessed using colony formation assays. Flow cytometry was used to evaluate cell apoptosis rate and the changes in cell cycle. Finally, apoptotic and cell cycle protein expression was immunodetected using western blot. RESULTS: Exposure to PARPi prior to standard chemotherapy sensitized BRCA1-mutated or epigenetically-silenced BRCA1 cell lines to lower doses of chemotherapy. Similar results were observed in BRCA1 wild-type and cell lines in which BRCA1 functionality was restored. Moreover, this treatment increased the apoptotic rate in these cell lines. CONCLUSION: Pre-treatment with PARPi followed by standard chemotherapy in vitro is more efficient in growth inhibition and induction of apoptosis compared to the administration of standard chemotherapy followed by PARPi.


Assuntos
Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína BRCA1/antagonistas & inibidores , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisplatino/farmacologia , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Terapia de Alvo Molecular , Mutação , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Reparo de DNA por Recombinação
14.
Nat Commun ; 10(1): 353, 2019 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-30664638

RESUMO

Mucosal melanoma is a rare and poorly characterized subtype of human melanoma. Here we perform a cross-species analysis by sequencing tumor-germline pairs from 46 primary human muscosal, 65 primary canine oral and 28 primary equine melanoma cases from mucosal sites. Analysis of these data reveals recurrently mutated driver genes shared between species such as NRAS, FAT4, PTPRJ, TP53 and PTEN, and pathogenic germline alleles of BRCA1, BRCA2 and TP53. We identify a UV mutation signature in a small number of samples, including human cases from the lip and nasal mucosa. A cross-species comparative analysis of recurrent copy number alterations identifies several candidate drivers including MDM2, B2M, KNSTRN and BUB1B. Comparison of somatic mutations in recurrences and metastases to those in the primary tumor suggests pervasive intra-tumor heterogeneity. Collectively, these studies suggest a convergence of some genetic changes in mucosal melanomas between species but also distinctly different paths to tumorigenesis.


Assuntos
Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica , Mutação em Linhagem Germinativa , Melanoma/genética , Neoplasias Bucais/genética , Proteínas de Neoplasias/genética , Neoplasias Cutâneas/genética , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Caderinas/genética , Caderinas/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Variações do Número de Cópias de DNA , Cães , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Cavalos , Humanos , Melanoma/metabolismo , Melanoma/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Membrana Mucosa/metabolismo , Membrana Mucosa/patologia , Proteínas de Neoplasias/metabolismo , Recidiva Local de Neoplasia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Especificidade da Espécie , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
15.
Nat Commun ; 10(1): 87, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30622252

RESUMO

Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation.


Assuntos
Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Reparo do DNA por Junção de Extremidades/genética , Resistencia a Medicamentos Antineoplásicos/genética , Animais , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Ligase Dependente de ATP/metabolismo , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Células-Tronco Embrionárias Murinas , Mutação , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Topotecan/farmacologia , Topotecan/uso terapêutico
16.
J Biochem Mol Toxicol ; 33(5): e22286, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30672063

RESUMO

The objective of the present study was to elucidate the effect of BMN 673 (talozoparib) on BRCA1 mutant (HCC1937) and wild-type (MDA-MB-231) triple negative breast cancer (TNBC). The in vitro cytotoxicity results indicated that BMN 673 had considerable inhibitory effects on HCC1937 and MDA-MB-231 cell lines by inducing apoptosis, multicaspase activity, G2/M arrest, and altering the expression levels of apoptosis-related genes (P < 0.01). Additionally, BMN 673 indicated no toxicity on MCF-10A control cells until a certain concentration and incubation time. However, BMN 673, a novel and selective poly ADP ribose polymerase inhibitor, was more potent in TNBC cells bearing BRCA1 mutant than those with wild-type BRCA1. In conclusion, our study, for the first time, demonstrated a molecular mechanism of the induction of apoptosis by BMN 673 in TNBC with different genetic profile. However, further investigations regarding the exact molecular mechanisms underlying BMN 673-inducing apoptotic death and gene-cell line associations are required.


Assuntos
Proteína BRCA1 , Mutação , Ftalazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias de Mama Triplo Negativas , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
17.
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
18.
Proc Natl Acad Sci U S A ; 116(2): 619-624, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30584090

RESUMO

Ovarian cancer remains the most lethal gynecologic malignancy. We analyzed the mutational landscape of 64 primary, 41 metastatic, and 17 recurrent fresh-frozen tumors from 77 patients along with matched normal DNA, by whole-exome sequencing (WES). We also sequenced 13 pairs of synchronous bilateral ovarian cancer (SBOC) to evaluate the evolutionary history. Lastly, to search for therapeutic targets, we evaluated the activity of the Bromodomain and Extra-Terminal motif (BET) inhibitor GS-626510 on primary tumors and xenografts harboring c-MYC amplifications. In line with previous studies, the large majority of germline and somatic mutations were found in BRCA1/2 (21%) and TP53 (86%) genes, respectively. Among mutations in known cancer driver genes, 77% were transmitted from primary tumors to metastatic tumors, and 80% from primary to recurrent tumors, indicating that driver mutations are commonly retained during ovarian cancer evolution. Importantly, the number, mutation spectra, and signatures in matched primary-metastatic tumors were extremely similar, suggesting transcoelomic metastases as an early dissemination process using preexisting metastatic ability rather than an evolution model. Similarly, comparison of SBOC showed extensive sharing of somatic mutations, unequivocally indicating a common ancestry in all cases. Among the 17 patients with matched tumors, four patients gained PIK3CA amplifications and two patients gained c-MYC amplifications in the recurrent tumors, with no loss of amplification or gain of deletions. Primary cell lines and xenografts derived from chemotherapy-resistant tumors demonstrated sensitivity to JQ1 and GS-626510 (P = 0.01), suggesting that oral BET inhibitors represent a class of personalized therapeutics in patients harboring recurrent/chemotherapy-resistant disease.


Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Mutação , Recidiva Local de Neoplasia , Proteínas , Proteínas Proto-Oncogênicas c-myc , Triazóis/farmacologia , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Feminino , Humanos , Camundongos , Metástase Neoplásica , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Neoplasias Ovarianas , Proteínas/antagonistas & inibidores , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
19.
DNA Repair (Amst) ; 74: 80-90, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30583959

RESUMO

The DNA double strand breaks (DSBs) created during meiotic recombination and during some types of chemotherapy contain protein covalently attached to their 5' termini. Removal of the end-blocking protein is a prerequisite to DSB processing by non-homologous end-joining or homologous recombination. One mechanism for removing the protein involves CtIP-stimulated Mre11-catalyzed nicking of the protein-linked strand distal to the DSB terminus, releasing the end-blocking protein while it remains covalently attached to an oligonucleotide. Much of what is known about this repair process has recently been deciphered through in vitro reconstitution studies. We present here a novel model system based on adenovirus (Ad), which contains the Ad terminal protein covalently linked to the 5' terminus of its dsDNA genome, for studying the repair of 5' protein-linked DSBs in vivo. It was previously shown that the genome of Ad mutants that lack early region 4 (E4) can be joined into concatemers in vivo, suggesting that the Ad terminal protein had been removed from the genome termini prior to ligation. Here we show that during infection with the E4-deleted Ad mutant dl1004, the Ad terminal protein is removed in a manner that recapitulates removal of end-blocking proteins from cellular DSBs. In addition to displaying a dependence on CtIP, and Mre11 acting as the endonuclease, the protein-linked oligonucleotides that are released from the viral genome are similar in size to the oligos that remain attached to Spo11 and Top2 after they are removed from the 5' termini of DSBs during meiotic recombination and etoposide chemotherapy, respectively. The single nucleotide resolution that is possible with this assay, combined with the single sequence context in which the lesion is presented, make it a useful tool for further refining our mechanistic understanding of how blocking proteins are removed from the 5' termini of DSBs.


Assuntos
Adenoviridae/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Genoma Viral/genética , Proteínas/metabolismo , Proteína BRCA1/metabolismo , Proteínas de Transporte/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/metabolismo
20.
Spectrochim Acta A Mol Biomol Spectrosc ; 209: 109-117, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30384016

RESUMO

SISLL-TAT and TAT-SISLL were synthesized by modifying the N- or C-termini of cell-penetrating peptides as transacting activator of transcription TAT (47-57) by attaching BRCA1 (782-786) (SISLL). The novel peptides were synthesized through Fmoc solid-phase synthesis procedures and characterized by LCQ Fleet MS, 1H NMR and 13C NMR. SISLL-TAT and TAT-SISLL displayed forceful antibacterial activities against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Salmonella typhimurium with low hemolysis. SISLL-TAT showed better antibacterial activity than TAT-SISLL, with the minimum inhibitory concentration (MIC) values of 10-33 µg·mL-1. The results of the DNA-binding activities showed that both SISLL-TAT and TAT-SISLL could interact with DNA via the minor groove mode, and the binding constants were 4.97 × 105 L·mol-1 and 4.42 × 105 L·mol-1 at 310 K, respectively. Circular dichroism analysis showed slight transformation of the lysozyme secondary structure caused by SISLL-TAT and TAT-SISLL. We also found that the novel peptides SISLL-TAT and TAT-SISLL targeted bacterial DNA resulting in cell death. This explains the antibacterial mechanism of SISLL-TAT and TAT-SISLL, and is a solid theoretical basis for further designing novel and highly effective antibiotics for clinical application.


Assuntos
Antibacterianos/farmacologia , Proteína BRCA1/química , Bactérias/efeitos dos fármacos , DNA Bacteriano/metabolismo , Muramidase/metabolismo , Fragmentos de Peptídeos/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Antibacterianos/química , Proteína BRCA1/metabolismo , Dicroísmo Circular , Humanos , Técnicas In Vitro , Testes de Sensibilidade Microbiana , Fragmentos de Peptídeos/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
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