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1.
PLoS Pathog ; 16(10): e1008849, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33002095

RESUMO

Epstein-Barr virus (EBV) causes lymphomas and epithelial cell cancers. Though generally silent in B lymphocytes, this widely prevalent virus can cause endemic Burkitt lymphoma and post-transplant lymphoproliferative disorders/lymphomas in immunocompromised hosts. By learning how EBV breaches barriers to cell proliferation, we hope to undermine those strategies to treat EBV lymphomas and potentially other cancers. We had previously found that EBV, through activation of cellular STAT3 prevents phosphorylation of Chk1, and thereby, suppresses activation of the intra-S phase cell-cycle checkpoint, a potent barrier to oncogene-driven proliferation. This observation prompted us to examine the consequences on DNA repair since homologous recombination repair, the most error-free form, requires phosphoChk1. We now report that the defect in Chk1 phosphorylation also curtails RAD51 nucleation, and thereby, homologous recombination repair of DNA double strand breaks. The resulting reliance on error-prone microhomology-mediated end-joining (MMEJ) repair makes EBV-transformed cells susceptible to PARP inhibition and simultaneous accrual of genome-wide deletions and insertions resulting from synthesis-dependent MMEJ. Analysis of transcriptomic and drug susceptibility data from hundreds of cancer lines reveals a STAT3-dependent gene-set predictive of susceptibility of cancers to synthetic lethal PARP inhibition. These findings i) demonstrate how the tumor virus EBV re-shapes cellular DNA repair, ii) provide the first genome-wide evidence for insertions resulting from MMEJ in human cells, and iii) expand the range of cancers (EBV-related and -unrelated) that are likely to respond to synthetic lethal inhibitors given the high prevalence of cancers with constitutively active STAT3.


Assuntos
Linfócitos B/virologia , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Quebras de DNA de Cadeia Dupla , Infecções por Vírus Epstein-Barr/virologia , Reparo de DNA por Recombinação , Fator de Transcrição STAT3/metabolismo , Adolescente , Adulto , Linfócitos B/citologia , Linfócitos B/metabolismo , Proteína BRCA1/genética , Proteína BRCA2/genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Linfoma de Burkitt/virologia , Proliferação de Células , Reparo do DNA por Junção de Extremidades , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/isolamento & purificação , Humanos , Neoplasias/genética , Neoplasias/patologia , Neoplasias/virologia , Fosforilação , Fator de Transcrição STAT3/genética , Adulto Jovem
2.
Oncogene ; 39(47): 7051-7062, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32989256

RESUMO

Multiple RNA processing events including transcription, mRNA splicing, and export are delicately coordinated by the TREX complex. As one of the essential subunits, DDX39B couples the splicing and export machineries by recruiting ALYREF onto mRNA. In this study, we further explore the functions of DDX39B in handling damaged DNA, and unexpectedly find that DDX39B facilitates DNA repair by homologous recombination through upregulating BRCA1. Specifically, DDX39B binds to and stabilizes BRCA1 mRNA. DDX39B ensures ssDNA formation and RAD51 accumulation at DSB sites by maintaining BRCA1 levels. Without DDX39B being present, ovarian cancer cells exhibit hypersensitivity to DNA-damaging chemotherapeutic agents like platinum or PARPi. Moreover, DDX39B-deficient mice show embryonic lethality or developmental retardation, highly reminiscent of those lacking BRCA1. High DDX39B expression is correlated with worse survival in ovarian cancer patients. Thus, DDX39B suppression represents a rational approach for enhancing the efficacy of chemotherapy in BRCA1-proficient ovarian cancers.


Assuntos
Antineoplásicos/farmacologia , Proteína BRCA1/genética , RNA Helicases DEAD-box/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Ovarianas/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Knockout , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Processamento de RNA/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação/efeitos dos fármacos , Taxa de Sobrevida , Fatores de Transcrição/metabolismo , Regulação para Cima
3.
Nat Commun ; 11(1): 4875, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978388

RESUMO

Single-cell whole-exome sequencing (scWES) is a powerful approach for deciphering intratumor heterogeneity and identifying cancer drivers. So far, however, simultaneous analysis of single nucleotide variants (SNVs) and copy number variations (CNVs) of a single cell has been challenging. By analyzing SNVs and CNVs simultaneously in bulk and single cells of premalignant tissues and tumors from mouse and human BRCA1-associated breast cancers, we discover an evolution process through which the tumors initiate from cells with SNVs affecting driver genes in the premalignant stage and malignantly progress later via CNVs acquired in chromosome regions with cancer driver genes. These events occur randomly and hit many putative cancer drivers besides p53 to generate unique genetic and pathological features for each tumor. Upon this, we finally identify a tumor metastasis suppressor Plekha5, whose deficiency promotes cancer metastasis to the liver and/or lung.


Assuntos
Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Predisposição Genética para Doença/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lesões Pré-Cancerosas/genética , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Heterogeneidade Genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fígado/patologia , Pulmão/patologia , Camundongos , Camundongos Knockout , Mutação , Lesões Pré-Cancerosas/patologia , Transcriptoma
4.
DNA Cell Biol ; 39(10): 1838-1849, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32876480

RESUMO

The breast cancer gene 1 (BRCA1) is a tumor suppressor, and mutations or epigenetic inactivation will increase the risk of breast cancer oncogenesis. The current research aimed to explore the relationship between BRCA1 expression, prognosis, and tumor immunity in hepatocellular carcinoma (HCC). In this study, BRCA1 expression was analyzed via multiple online databases and its association with clinical characteristics, prognosis and genetic alterations was identified using the original The Cancer Genome Atlas-liver hepatocellular carcinoma cohorts. DNA methylation sites and their prognostic values were analyzed using MethSurv. The correlations between BRCA1 and immune infiltration were investigated via Tumor Immune Estimation Resource. As results, BRCA1 was significantly upregulated in tumor tissues in multiple HCC cohorts. Besides, high BRCA1 expression was correlated with race, advanced T stage, clinical stage, poor tumor grade, MSI status, and worse prognosis. Notably, BRCA1 expression was positively correlated with infiltration levels of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells. The current findings imply that BRCA1 is associated with prognosis and immune infiltration, laying foundations for in-depth research on the role of BRCA1 in HCC.


Assuntos
Proteína BRCA1/genética , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteína BRCA1/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Movimento Celular , Metilação de DNA , Células Dendríticas/imunologia , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Linfócitos/imunologia , Macrófagos/imunologia
5.
Mol Cell ; 80(2): 327-344.e8, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32966758

RESUMO

Stabilization of stalled replication forks is a prominent mechanism of PARP (Poly(ADP-ribose) Polymerase) inhibitor (PARPi) resistance in BRCA-deficient tumors. Epigenetic mechanisms of replication fork stability are emerging but remain poorly understood. Here, we report the histone acetyltransferase PCAF (p300/CBP-associated) as a fork-associated protein that promotes fork degradation in BRCA-deficient cells by acetylating H4K8 at stalled replication forks, which recruits MRE11 and EXO1. A H4K8ac binding domain within MRE11/EXO1 is required for their recruitment to stalled forks. Low PCAF levels, which we identify in a subset of BRCA2-deficient tumors, stabilize stalled forks, resulting in PARPi resistance in BRCA-deficient cells. Furthermore, PCAF activity is tightly regulated by ATR (ataxia telangiectasia and Rad3-related), which phosphorylates PCAF on serine 264 (S264) to limit its association and activity at stalled forks. Our results reveal PCAF and histone acetylation as critical regulators of fork stability and PARPi responses in BRCA-deficient cells, which provides key insights into targeting BRCA-deficient tumors and identifying epigenetic modulators of chemotherapeutic responses.


Assuntos
Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA , Exodesoxirribonucleases/metabolismo , Histonas/metabolismo , Proteína Homóloga a MRE11/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação/efeitos dos fármacos , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Replicação do DNA/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lisina/metabolismo , Modelos Biológicos , Mutação/genética , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ligação Proteica/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/química , Fatores de Transcrição de p300-CBP/genética
6.
Am J Hum Genet ; 107(4): 654-669, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32937144

RESUMO

There is growing recognition that epivariations, most often recognized as promoter hypermethylation events that lead to gene silencing, are associated with a number of human diseases. However, little information exists on the prevalence and distribution of rare epigenetic variation in the human population. In order to address this, we performed a survey of methylation profiles from 23,116 individuals using the Illumina 450k array. Using a robust outlier approach, we identified 4,452 unique autosomal epivariations, including potentially inactivating promoter methylation events at 384 genes linked to human disease. For example, we observed promoter hypermethylation of BRCA1 and LDLR at population frequencies of ∼1 in 3,000 and ∼1 in 6,000, respectively, suggesting that epivariations may underlie a fraction of human disease which would be missed by purely sequence-based approaches. Using expression data, we confirmed that many epivariations are associated with outlier gene expression. Analysis of variation data and monozygous twin pairs suggests that approximately two-thirds of epivariations segregate in the population secondary to underlying sequence mutations, while one-third are likely sporadic events that occur post-zygotically. We identified 25 loci where rare hypermethylation coincided with the presence of an unstable CGG tandem repeat, validated the presence of CGG expansions at several loci, and identified the putative molecular defect underlying most of the known folate-sensitive fragile sites in the genome. Our study provides a catalog of rare epigenetic changes in the human genome, gives insight into the underlying origins and consequences of epivariations, and identifies many hypermethylated CGG repeat expansions.


Assuntos
Proteína BRCA1/genética , Epigênese Genética , Doenças Genéticas Inatas/genética , Genoma Humano , Receptores de LDL/genética , Expansão das Repetições de Trinucleotídeos , Proteína BRCA1/metabolismo , Metilação de DNA , Feminino , Ácido Fólico/metabolismo , Inativação Gênica , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/patologia , Loci Gênicos , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Regiões Promotoras Genéticas , Receptores de LDL/metabolismo , Gêmeos Monozigóticos
7.
Sci Rep ; 10(1): 13275, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764667

RESUMO

The therapeutic effect of irradiation is thought to come from DNA damage that affects rapidly proliferating cancer cells; however, resistant cells rapidly initiate mechanisms to repair such damage. While DNA repair mechanisms responsible for cancer cell survival following DNA damage are understood, less is known about the epigenetic mechanisms resulting in resistance to radiotherapy. Although changes in DNA methylation are related to mechanisms of long-term resistance, it is more likely that the methylation state of a series of proteins could be responsible for the first-line of defense of cancer cells against irradiation. In this study, we observed that irradiation of breast cancer cells was accompanied by an overproduction in S-adenosylmethionine, which increases the activity of cellular methylases. We found that by activating PRMT1, irradiation triggers a BRCA1-dependent program that results in efficient DNA repair and inhibition of apoptosis. Depletion of PRMT1 in irradiated cells resulted in a switch of BRCA1 functions from repair and survival in the nucleus to activation of cell death signals in the cytoplasm. We conclude that by modulating the cellular localization of BRCA1, PRMT1 is an important regulator of the oncogenic functions of BRCA1, contributing to the epigenetic defense of breast cancer cells against ionizing radiation.


Assuntos
Proteína BRCA1/metabolismo , Neoplasias da Mama/patologia , Proteína-Arginina N-Metiltransferases/metabolismo , Tolerância a Radiação , Proteínas Repressoras/metabolismo , S-Adenosilmetionina/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular , Citoplasma/metabolismo , Reparo do DNA , Epigênese Genética , Feminino , Humanos , Células MCF-7 , Camundongos , Radiação Ionizante , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Virology ; 548: 174-181, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838940

RESUMO

The incidence of head and neck squamous cell carcinomas (HNSCCs) is rising in developed countries. This is driven by an increase in HNSCCs caused by high-risk human papillomavirus (HPV) infections or HPV + HNSCCs. Compared to HNSCCs not caused by HPV (HPV- HNSCCs), HPV + HNSCCs are more responsive to therapy and associated with better oncologic outcomes. As a result, the HPV status of an HNSCC is an important determinant in medical management. One method to determine the HPV status of an HNSCC is increased expression of p16 caused by the HPV E7 oncogene. We identified novel expression changes in HPV + HNSCCs. A comparison of gene expression among HPV+ and HPV- HNSCCs in The Cancer Genome Atlas demonstrated increased DNA repair gene expression in HPV + HNSCCs. Further, DNA repair gene expression correlated with HNSCC survival. Immunohistochemical analysis of a novel HNSCC microarray confirmed that DNA repair protein abundance is elevated in HPV + HNSCCs.


Assuntos
Alphapapillomavirus/metabolismo , Infecções por Papillomavirus/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adulto , Idoso , Alphapapillomavirus/genética , Alphapapillomavirus/isolamento & purificação , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Reparo do DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
9.
PLoS One ; 15(7): e0235766, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639993

RESUMO

High-grade serous ovarian carcinoma (HGSOC) remains the deadliest form of epithelial ovarian cancer and despite major efforts little improvement in overall survival has been achieved. Identification of recurring "driver" genetic lesions has the potential to enable design of novel therapies for cancer. Here, we report on a study to find such new therapeutic targets for HGSOC using exome-capture sequencing approach targeting all kinase genes in 127 patient samples. Consistent with previous reports, the most frequently mutated gene was TP53 (97% mutation frequency) followed by BRCA1 (10% mutation frequency). The average mutation frequency of the kinase genes mutated from our panel was 1.5%. Intriguingly, after BRCA1, JAK3 was the most frequently mutated gene (4% mutation frequency). We tested the transforming properties of JAK3 mutants using the Ba/F3 cell-based in vitro functional assay and identified a novel gain-of-function mutation in the kinase domain of JAK3 (p.T1022I). Importantly, p.T1022I JAK3 mutants displayed higher sensitivity to the JAK3-selective inhibitor Tofacitinib compared to controls. For independent validation, we re-sequenced the entire JAK3 coding sequence using tagged amplicon sequencing (TAm-Seq) in 463 HGSOCs resulting in an overall somatic mutation frequency of 1%. TAm-Seq screening of CDK12 in the same population revealed a 7% mutation frequency. Our data confirms that the frequency of mutations in kinase genes in HGSOC is low and provides accurate estimates for the frequency of JAK3 and CDK12 mutations in a large well characterized cohort. Although p.T1022I JAK3 mutations are rare, our functional validation shows that if detected they should be considered as potentially actionable for therapy. The observation of CDK12 mutations in 7% of HGSOC cases provides a strong rationale for routine somatic testing, although more functional and clinical characterization is required to understand which nonsynonymous mutations alterations are associated with homologous recombination deficiency.


Assuntos
Proteína BRCA1/genética , Cistadenocarcinoma Seroso/genética , Janus Quinase 3/genética , Mutação , Neoplasias Ovarianas/genética , Proteínas Quinases/genética , Proteína Supressora de Tumor p53/genética , Proteína BRCA1/metabolismo , Estudos de Casos e Controles , Estudos de Coortes , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Janus Quinase 3/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo
10.
Nat Commun ; 11(1): 3296, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620799

RESUMO

Identifying robust, patient-specific, and predictive biomarkers presents a major obstacle in precision oncology. To optimize patient-specific therapeutic strategies, here we couple pathway knowledge with large-scale drug sensitivity, RNAi, and CRISPR-Cas9 screening data from 460 cell lines. Pathway activity levels are found to be strong predictive biomarkers for the essentiality of 15 proteins, including the essentiality of MAD2L1 in breast cancer patients with high BRCA-pathway activity. We also find strong predictive biomarkers for the sensitivity to 31 compounds, including BCL2 and microtubule inhibitors (MTIs). Lastly, we show that Bcl-xL inhibition can modulate the activity of a predictive biomarker pathway and re-sensitize lung cancer cells and tumors to MTI therapy. Overall, our results support the use of pathways in helping to achieve the goal of precision medicine by uncovering dozens of predictive biomarkers.


Assuntos
Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Transdução de Sinais/genética , Animais , Antineoplásicos/farmacologia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Redes Reguladoras de Genes , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Medicina de Precisão/métodos , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
11.
Nat Commun ; 11(1): 3726, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709856

RESUMO

Ovarian cancer (OVCA) inevitably acquires resistance to platinum chemotherapy and PARP inhibitors (PARPi). We show that acquisition of PARPi-resistance is accompanied by increased ATR-CHK1 activity and sensitivity to ATR inhibition (ATRi). However, PARPi-resistant cells are remarkably more sensitive to ATRi when combined with PARPi (PARPi-ATRi). Sensitivity to PARPi-ATRi in diverse PARPi and platinum-resistant models, including BRCA1/2 reversion and CCNE1-amplified models, correlate with synergistic increases in replication fork stalling, double-strand breaks, and apoptosis. Surprisingly, BRCA reversion mutations and an ability to form RAD51 foci are frequently not observed in models of acquired PARPi-resistance, suggesting the existence of alternative resistance mechanisms. However, regardless of the mechanisms of resistance, complete and durable therapeutic responses to PARPi-ATRi that significantly increase survival are observed in clinically relevant platinum and acquired PARPi-resistant patient-derived xenografts (PDXs) models. These findings indicate that PARPi-ATRi is a highly promising strategy for OVCAs that acquire resistance to PARPi and platinum.


Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Platina/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Ciclinas/metabolismo , Combinação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Técnicas de Inativação de Genes , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Ovarianas/genética , Rad51 Recombinase/metabolismo , Células-Tronco , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Cancer Res ; 80(14): 2977-2978, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669350

RESUMO

Cancer cells with germline deleterious mutations of BRCA1 or BRCA2 are deficient in homologous recombination repair and therefore sensitive to PARP inhibitor treatment. However, wild-type BRCA1/2-expressing cells with defects in other DNA damage repair pathway components may also exhibit "BRCAness," which in combination with PARP inhibition can similarly induce synthetic lethality. In this issue of Cancer Research, Luo and colleagues report a novel mechanism by which BRCA1 protein degradation in response to DNA double-strand breaks is regulated by prolyl isomerase Pin1. Inactivation of Pin1 can establish BRCAness in cancer cells and thus sensitize cells to PARP inhibitor treatment.See related articles by Luo et al., p. 3033.


Assuntos
Neoplasias da Mama , Inibidores de Poli(ADP-Ribose) Polimerases , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Neoplasias da Mama/tratamento farmacológico , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Humanos , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico
13.
Mol Cell ; 79(3): 425-442.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32615088

RESUMO

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.


Assuntos
Adenosina/análogos & derivados , Neoplasias de Cabeça e Pescoço/genética , Metiltransferases/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adenosina/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Bleomicina/farmacologia , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Células HEK293 , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/metabolismo , Hibridização de Ácido Nucleico , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Nat Commun ; 11(1): 3256, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591500

RESUMO

BRCA1 mutation carriers have a higher risk of developing triple-negative breast cancer (TNBC), which is a refractory disease due to its non-responsiveness to current clinical targeted therapies. Using the Sleeping Beauty transposon system in Brca1-deficient mice, we identified 169 putative cancer drivers, among which Notch1 is a top candidate for accelerating TNBC by promoting the epithelial-mesenchymal transition (EMT) and regulating the cell cycle. Activation of NOTCH1 suppresses mitotic catastrophe caused by BRCA1 deficiency by restoring S/G2 and G2/M cell cycle checkpoints, which may through activation of ATR-CHK1 signalling pathway. Consistently, analysis of human breast cancer tissue demonstrates NOTCH1 is highly expressed in TNBCs, and the activated form of NOTCH1 correlates positively with increased phosphorylation of ATR. Additionally, we demonstrate that inhibition of the NOTCH1-ATR-CHK1 cascade together with cisplatin synergistically kills TNBC by targeting the cell cycle checkpoint, DNA damage and EMT, providing a potent clinical option for this fatal disease.


Assuntos
Proteína BRCA1/deficiência , Carcinogênese/patologia , Receptor Notch1/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Alelos , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Morte Celular , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Elementos de DNA Transponíveis/genética , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Knockout , Mitose , Mutação/genética , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/genética
15.
Mol Cell Proteomics ; 19(9): 1503-1522, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32591346

RESUMO

As the COVID-19 pandemic continues to spread, thousands of scientists around the globe have changed research direction to understand better how the virus works and to find out how it may be tackled. The number of manuscripts on preprint servers is soaring and peer-reviewed publications using MS-based proteomics are beginning to emerge. To facilitate proteomic research on SARS-CoV-2, the virus that causes COVID-19, this report presents deep-scale proteomes (10,000 proteins; >130,000 peptides) of common cell line models, notably Vero E6, Calu-3, Caco-2, and ACE2-A549 that characterize their protein expression profiles including viral entry factors such as ACE2 or TMPRSS2. Using the 9 kDa protein SRP9 and the breast cancer oncogene BRCA1 as examples, we show how the proteome expression data can be used to refine the annotation of protein-coding regions of the African green monkey and the Vero cell line genomes. Monitoring changes of the proteome on viral infection revealed widespread expression changes including transcriptional regulators, protease inhibitors, and proteins involved in innate immunity. Based on a library of 98 stable-isotope labeled synthetic peptides representing 11 SARS-CoV-2 proteins, we developed PRM (parallel reaction monitoring) assays for nano-flow and micro-flow LC-MS/MS. We assessed the merits of these PRM assays using supernatants of virus-infected Vero E6 cells and challenged the assays by analyzing two diagnostic cohorts of 24 (+30) SARS-CoV-2 positive and 28 (+9) negative cases. In light of the results obtained and including recent publications or manuscripts on preprint servers, we critically discuss the merits of MS-based proteomics for SARS-CoV-2 research and testing.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/genética , Interações Hospedeiro-Patógeno/genética , Pneumonia Viral/genética , Proteômica/métodos , Proteínas Virais/genética , Células A549 , Sequência de Aminoácidos , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Betacoronavirus/patogenicidade , Células CACO-2 , Estudos de Casos e Controles , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Regulação da Expressão Gênica , Ontologia Genética , Humanos , Indicadores e Reagentes , Anotação de Sequência Molecular , Fases de Leitura Aberta , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Proteômica/instrumentação , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/metabolismo , Transdução de Sinais , Células Vero , Proteínas Virais/classificação , Proteínas Virais/metabolismo , Internalização do Vírus
16.
Mol Cell ; 78(6): 1152-1165.e8, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32516598

RESUMO

The APEX2 gene encodes APE2, a nuclease related to APE1, the apurinic/apyrimidinic endonuclease acting in base excision repair. Loss of APE2 is lethal in cells with mutated BRCA1 or BRCA2, making APE2 a prime target for homologous recombination-defective cancers. However, because the function of APE2 in DNA repair is poorly understood, it is unclear why BRCA-deficient cells require APE2 for viability. Here we present the genetic interaction profiles of APE2, APE1, and TDP1 deficiency coupled to biochemical and structural dissection of APE2. We conclude that the main role of APE2 is to reverse blocked 3' DNA ends, problematic lesions that preclude DNA synthesis. Our work also suggests that TOP1 processing of genomic ribonucleotides is the main source of 3'-blocking lesions relevant to APEX2-BRCA1/2 synthetic lethality. The exquisite sensitivity of BRCA-deficient cells to 3' blocks indicates that they represent a tractable vulnerability in homologous recombination-deficient tumor cells.


Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Enzimas Multifuncionais/metabolismo , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular , DNA/metabolismo , Dano ao DNA , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases/genética , Genes BRCA1/fisiologia , Humanos , Enzimas Multifuncionais/genética , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo
17.
Nucleic Acids Res ; 48(11): 6032-6052, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427332

RESUMO

Chromosomal double strand breaks (DSBs) can initiate several signaling events, such as ubiquitination, however the precise influence of such signaling on DSB repair outcomes remains poorly understood. With an RNA interference screen, we found that the E3 ubiquitin ligase RNF8 suppresses a deletion rearrangement mediated by canonical non-homologous end joining (C-NHEJ). We also found that RNF8 suppresses EJ without insertion/deletion mutations, which is a hallmark of C-NHEJ. Conversely, RNF8 promotes alternative EJ (ALT-EJ) events involving microhomology that is embedded from the edge of the DSB. These ALT-EJ events likely require limited end resection, whereas RNF8 is not required for single-strand annealing repair involving extensive end resection. Thus, RNF8 appears to specifically facilitate repair events requiring limited end resection, which we find is dependent on the DSB end protection factor KU. However, we also find that RNF8 is important for homology-directed repair (HDR) independently of KU, which appears linked to promoting PALB2 function. Finally, the influence of RNF8 on EJ is distinct from 53BP1 and the ALT-EJ factor, POLQ. We suggest that RNF8 mediates both ALT-EJ and HDR, but via distinct mechanisms, since only the former is dependent on KU.


Assuntos
Quebra Cromossômica , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Autoantígeno Ku/metabolismo , Reparo de DNA por Recombinação , Ubiquitina-Proteína Ligases/metabolismo , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Ligação a DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Humanos , Mutação INDEL , Proteínas Nucleares/metabolismo , Domínios Proteicos , Interferência de RNA , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação/genética , Deleção de Sequência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/química
18.
Mol Cell ; 78(5): 951-959.e6, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32359443

RESUMO

BRCA1 promotes the DNA end resection and RAD51 loading steps of homologous recombination (HR). Whether these functions can be uncoupled, and whether mutant proteins retaining partial activity can complement one another, is unclear and could affect the severity of BRCA1-associated Fanconi anemia (FA). Here we generated a Brca1CC mouse with a coiled-coil (CC) domain deletion. Brca1CC/CC mice are born at low frequencies, and post-natal mice have FA-like abnormalities, including bone marrow failure. Intercrossing with Brca1Δ11, which is homozygous lethal, generated Brca1CC/Δ11 mice at Mendelian frequencies that were indistinguishable from Brca1+/+ mice. Brca1CC and Brca1Δ11 proteins were individually responsible for counteracting 53BP1-RIF1-Shieldin activity and promoting RAD51 loading, respectively. Thus, Brca1CC and Brca1Δ11 alleles represent separation-of-function mutations that combine to provide a level of HR sufficient for normal development and hematopoiesis. Because BRCA1 activities can be genetically separated, compound heterozygosity for functional complementary mutations may protect individuals from FA.


Assuntos
Proteína BRCA1/genética , Recombinação Homóloga/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Proteína BRCA1/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Éxons , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
19.
J Biomed Sci ; 27(1): 61, 2020 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-32381096

RESUMO

BACKGROUND: The disruption of the blood-brain barrier (BBB) plays a critical event in the pathogenesis of ischemia stroke. TGR5 is recognized as a potential target for the treatment for neurologic disorders. METHODS: This study investigated the roles of TGR5 activation in attenuating BBB damage and underlying mechanisms after middle cerebral artery occlusion (MCAO). Sprague-Dawley rats were subjected to model of MCAO and TGR5 agonist, INT777, was administered intranasally. Small interfering RNA (siRNA) for TGR5 and BRCA1 were administered through intracerebroventricular injection 48 h before MCAO. Infarct volumes, brain water content, BBB permeability, neurological scores, Western blot, immunofluorescence staining and co- immunoprecipitation were evaluated. RESULTS: Endogenous TGR5 and BRCA1 were upregulated in the injured hemisphere after MCAO and TGR5 expressed in endothelial cells. Treatment with INT777 alleviated brain water content and BBB permeability, reduced infarction volume and improved neurological scores at 24 h and 72 h after ischemia. INT777 administration increased BRCA1 and Sirt1 expression, as well as upregulated expressions of tight junction proteins. Ischemic damage induced interaction of TGR5 with BRCA1. TGR5 siRNA and BRCA1 siRNA significantly inhibited expressions of BRCA1 and Sirt1, aggravated BBB permeability and exacerbated stroke outcomes after MCAO. The protective effects of INT777 at 24 h after MCAO were also abolished by TGR5 siRNA or BRCA1 siRNA. CONCLUSIONS: Our findings demonstrate that activating TGR5 could reduce BBB breakdown and improve neurological functions through BRCA1/Sirt1 signaling pathway after MCAO. TGR5 may serve as a potential new candidate to relieve brain injury after MCAO.


Assuntos
Barreira Hematoencefálica/fisiologia , Infarto da Artéria Cerebral Média/patologia , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais/genética , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo
20.
Nucleic Acids Res ; 48(10): 5485-5498, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32347940

RESUMO

DNA double-strand breaks are repaired by end-joining or homologous recombination. A key-committing step of recombination is DNA end resection. In resection, phosphorylated CtIP first promotes the endonuclease of MRE11-RAD50-NBS1 (MRN). Subsequently, CtIP also stimulates the WRN/BLM-DNA2 pathway, coordinating thus both short and long-range resection. The structure of CtIP differs from its orthologues in yeast, as it contains a large internal unstructured region. Here, we conducted a domain analysis of CtIP to define the function of the internal region in DNA end resection. We found that residues 350-600 were entirely dispensable for resection in vitro. A mutant lacking these residues was unexpectedly more efficient than full-length CtIP in DNA end resection and homologous recombination in vivo, and consequently conferred resistance to lesions induced by the topoisomerase poison camptothecin, which require high MRN-CtIP-dependent resection activity for repair. This suggested that the internal CtIP region, further mapped to residues 550-600, may mediate a negative regulatory function to prevent over resection in vivo. The CtIP internal deletion mutant exhibited sensitivity to other DNA-damaging drugs, showing that upregulated resection may be instead toxic under different conditions. These experiments together identify a region within the central CtIP domain that negatively regulates DNA end resection.


Assuntos
Reparo do DNA , Endodesoxirribonucleases/química , Endodesoxirribonucleases/fisiologia , Proteína BRCA1/metabolismo , Camptotecina/toxicidade , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Endodesoxirribonucleases/genética , Humanos , Domínios Proteicos , Deleção de Sequência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
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