RESUMO
O-linked ß-N-acetyl glucosamine (O-GlcNAc) is attached to proteins under glucose-replete conditions; this posttranslational modification results in molecular and physiological changes that affect cell fate. Here we show that posttranslational modification of serine/arginine-rich protein kinase 2 (SRPK2) by O-GlcNAc regulates de novo lipogenesis by regulating pre-mRNA splicing. We found that O-GlcNAc transferase O-GlcNAcylated SRPK2 at a nuclear localization signal (NLS), which triggers binding of SRPK2 to importin α. Consequently, O-GlcNAcylated SRPK2 was imported into the nucleus, where it phosphorylated serine/arginine-rich proteins and promoted splicing of lipogenic pre-mRNAs. We determined that protein nuclear import by O-GlcNAcylation-dependent binding of cargo protein to importin α might be a general mechanism in cells. This work reveals a role of O-GlcNAc in posttranscriptional regulation of de novo lipogenesis, and our findings indicate that importin α is a "reader" of an O-GlcNAcylated NLS.
Assuntos
Neoplasias da Mama/metabolismo , Glucose/metabolismo , Lipogênese , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Neoplasias da Mama/genética , Proliferação de Células , Feminino , Glicosilação , Células HEK293 , Humanos , Células MCF-7 , Camundongos Nus , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Carga Tumoral , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
Long non-coding RNAs (lncRNAs) are emerging regulators of genomic stability and human disease. However, the molecular mechanisms by which nuclear lncRNAs directly contribute to DNA damage responses remain largely unknown. Using RNA antisense purification coupled with quantitative mass spectrometry (RAP-qMS), we found that the lncRNA BGL3 binds to PARP1 and BARD1, exhibiting unexpected roles in homologous recombination. Mechanistically, BGL3 is recruited to DNA double-strand breaks (DSBs) by PARP1 at an early time point, which requires its interaction with the DNA-binding domain of PARP1. BGL3 also binds the C-terminal BRCT domain and an internal region (amino acids 127-424) of BARD1, which mediates interaction of the BRCA1/BARD1 complex with its binding partners such as HP1γ and RAD51, resulting in BRCA1/BARD1 retention at DSBs. Cells depleted for BGL3 displayed genomic instability and were sensitive to DNA-damaging reagents. Overall, our findings underscore the biochemical versatility of RNA as a mediator molecule in the DNA damage response pathway, which affects the accumulation of BRCA1/BARD1 at DSBs.
Assuntos
Proteína BRCA1/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Complexos Multiproteicos/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína BRCA1/genética , Células HEK293 , Humanos , Células MCF-7 , Complexos Multiproteicos/genética , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Domínios Proteicos , RNA Longo não Codificante/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The replisome is important for DNA replication checkpoint activation, but how specific components of the replisome coordinate with ATR to activate Chk1 in human cells remains largely unknown. Here, we demonstrate that And-1, a replisome component, acts together with ATR to activate Chk1. And-1 is phosphorylated at T826 by ATR following replication stress, and this phosphorylation is required for And-1 to accumulate at the damage sites, where And-1 promotes the interaction between Claspin and Chk1, thereby stimulating efficient Chk1 activation by ATR. Significantly, And-1 binds directly to ssDNA and facilitates the association of Claspin with ssDNA. Furthermore, And-1 associates with replication forks and is required for the recovery of stalled forks. These studies establish a novel ATR-And-1 axis as an important regulator for efficient Chk1 activation and reveal a novel mechanism of how the replisome regulates the replication checkpoint and genomic stability.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Modelos Biológicos , Proteínas Quinases/metabolismo , Anticorpos/imunologia , Quinase 1 do Ponto de Checagem , Imunofluorescência , Células HEK293 , Humanos , Imunoprecipitação , Espectrometria de Massas , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Homologous recombination (HR) is a major mechanism to repair DNA double-strand breaks (DSBs). Although tumor suppressor CtIP is critical for DSB end resection, a key initial event of HR repair, the mechanism regulating the recruitment of CtIP to DSB sites remains largely unknown. Here, we show that acidic nucleoplasmic DNA-binding protein 1 (And-1) forms complexes with CtIP as well as other repair proteins, and is essential for HR repair by regulating DSB end resection. Furthermore, And-1 is recruited to DNA DSB sites in a manner dependent on MDC1, BRCA1 and ATM, down-regulation of And-1 impairs end resection by reducing the recruitment of CtIP to damage sites, and considerably reduces Chk1 activation and other damage response during HR repair. These findings collectively demonstrate a hitherto unknown role of MDC1âAnd-1âCtIP axis that regulates CtIP-mediated DNA end resection and cellular response to DSBs.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Reparo de DNA por Recombinação , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Endodesoxirribonucleases , Humanos , Proteínas Nucleares/metabolismoRESUMO
In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutantcas003 with nonsense mutation in topbp1 gene encoding topoisomerase II ß binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.
Assuntos
Proteínas de Transporte/genética , Sobrevivência Celular/genética , Reparo do DNA/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Proteínas de Peixe-Zebra/genética , Animais , Apoptose/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Transporte/metabolismo , Movimento Celular/genética , Proliferação de Células , Quinase 1 do Ponto de Checagem , Códon sem Sentido/genética , Dano ao DNA/genética , Replicação do DNA/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Embrião não Mamífero/metabolismo , Ativação Enzimática/genética , Células-Tronco Hematopoéticas/metabolismo , Hidroxiureia/farmacologia , Proteínas Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
This paper presents a new optical fiber distributed sensing concept based on coherent microwave-photonics interferometry (CMPI), which uses a microwave modulated coherent light source to interrogate cascaded interferometers for distributed measurement. By scanning the microwave frequencies, the complex microwave spectrum is obtained and converted to time domain signals at known locations by complex Fourier transform. The amplitudes of these time domain pulses are a function of the optical path differences (OPDs) of the distributed interferometers. Cascaded fiber Fabry-Perot interferometers (FPIs) fabricated by femtosecond laser micromachining were used to demonstrate the concept. The experimental results indicated that the strain measurement resolution can be better than 0.6 µÎµ using a FPI with a cavity length of 1.5 cm. Further improvement of the strain resolution to the nε level is achievable by increasing the cavity length of the FPI to over 1m. The tradeoff between the sensitivity and dynamic range was also analyzed in detail. To minimize the optical power instability (either from the light source or the fiber loss) induced errors, a single reflector was added in front of an individual FPI as an optical power reference for the purpose of compensation.
RESUMO
We report an open cavity, cantilever-based fiber optic Fabry-Perot interferometer hydrophone. The hydrophone is made of fused silica material, and its micro-cantilever beam is directly fabricated by femtosecond (fs) laser micromachining. The theoretical analyses and experimental verifications of the frequency response of the sensor are presented.
RESUMO
The centromere is an epigenetically designated chromatin domain that is essential for the accurate segregation of chromosomes during mitosis. The incorporation of centromere protein A (CENP-A) into chromatin is fundamental in defining the centromeric loci. Newly synthesized CENP-A is loaded at centromeres in early G(1) phase by the CENP-A-specific histone chaperone Holliday junction recognition protein (HJURP) coupled with other chromatin assembly factors. However, it is unknown whether there are additional HJURP-interacting factor(s) involving in this process. Here we identify acidic nucleoplasmic DNA-binding protein 1 (And-1) as a new factor that is required for the assembly of CENP-A nucleosomes. And-1 interacts with both CENP-A and HJURP in a prenucleosomal complex, and the association of And-1 with CENP-A is increased during the cell cycle transition from mitosis to G(1) phase. And-1 down-regulation significantly compromises chromosome congression and the deposition of HJURP-CENP-A complexes at centromeres. Consistently, overexpression of And-1 enhances the assembly of CENP-A at centromeres. We conclude that And-1 is an important factor that functions together with HJURP to facilitate the cell cycle-specific recruitment of CENP-A to centromeres.
Assuntos
Autoantígenos/genética , Centrômero/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , Cromossomos Humanos/metabolismo , Proteínas de Ligação a DNA/genética , Autoantígenos/metabolismo , Linhagem Celular Tumoral , Centrômero/química , Proteína Centromérica A , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Cromossomos Humanos/química , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Genes Reporter , Humanos , Luciferases , Mitose , Ligação Proteica , RNA Interferente Pequeno/genéticaRESUMO
Cyclin-dependent kinases 4 and 6 (CDK4/6) play a pivotal role in cell cycle and cancer development. Targeting CDK4/6 has demonstrated promising effects against breast cancer. However, resistance to CDK4/6 inhibitors (CDK4/6i), such as palbociclib, remains a substantial challenge in clinical settings. Using high-throughput combinatorial drug screening and genomic sequencing, we find that the microphthalmia-associated transcription factor (MITF) is activated via O-GlcNAcylation by O-GlcNAc transferase (OGT) in palbociclib-resistant breast cancer cells and tumors. Mechanistically, O-GlcNAcylation of MITF at Serine 49 enhances its interaction with importin α/ß, thus promoting its translocation to nuclei, where it suppresses palbociclib-induced senescence. Inhibition of MITF or its O-GlcNAcylation re-sensitizes resistant cells to palbociclib. Moreover, clinical studies confirm the activation of MITF in tumors from patients who are palbociclib-resistant or undergoing palbociclib treatment. Collectively, our studies shed light on the mechanism regulating palbociclib resistance and present clinical evidence for developing therapeutic approaches to treat CDK4/6i-resistant breast cancer patients.
Assuntos
Neoplasias da Mama , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Resistencia a Medicamentos Antineoplásicos , Fator de Transcrição Associado à Microftalmia , N-Acetilglucosaminiltransferases , Piperazinas , Piridinas , Humanos , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Neoplasias da Mama/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Quinase 6 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Fator de Transcrição Associado à Microftalmia/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Feminino , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Piperazinas/farmacologia , Piridinas/farmacologia , Linhagem Celular Tumoral , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/genética , Animais , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
DNA replication in all eukaryotes starts with the process of loading the replicative helicase MCM2-7 onto chromatin during late mitosis of the cell cycle. MCM2-7 is a key component of the prereplicative complex (pre-RC), which is loaded onto chromatin by the concerted action of origin recognition complex, Cdc6, and Cdt1. Here, we demonstrate that And-1 is assembled onto chromatin in late mitosis and early G(1) phase before the assembly of pre-RC in human cells. And-1 forms complexes with MCM2-7 to facilitate the assembly of MCM2-7 onto chromatin at replication origins in late mitosis and G(1) phase. We also present data to show that depletion of And-1 significantly reduces the interaction between Cdt1 and MCM7 in G(1) phase cells. Thus, human And-1 facilitates loading of the MCM2-7 helicase onto chromatin during the assembly of pre-RC.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Acetilação , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatina/metabolismo , Proteínas de Ligação a DNA/química , Fase G1 , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Componente 2 do Complexo de Manutenção de Minicromossomo , Componente 7 do Complexo de Manutenção de Minicromossomo , Proteínas Nucleares/metabolismo , Ligação Proteica , Origem de Replicação , TelófaseRESUMO
High-throughput screening (HTS) provides a rapid and comprehensive approach to identifying compounds that target specific biological processes as well as genes that are essential to those processes. Here we describe a HTS assay for small molecules that induce either DNA re-replication or endoreduplication (i.e. excess DNA replication) selectively in cells derived from human cancers. Such molecules will be useful not only to investigate cell division and differentiation, but they may provide a novel approach to cancer chemotherapy. Since induction of DNA re-replication results in apoptosis, compounds that selectively induce DNA re-replication in cancer cells without doing so in normal cells could kill cancers in vivo without preventing normal cell proliferation. Furthermore, the same HTS assay can be adapted to screen siRNA molecules to identify genes whose products restrict genome duplication to once per cell division. Some of these genes might regulate the formation of terminally differentiated polyploid cells during normal human development, whereas others will prevent DNA re-replication during each cell division. Based on previous studies, we anticipate that one or more of the latter genes will prove to be essential for proliferation of cancer cells but not for normal cells, since many cancer cells are deficient in mechanisms that maintain genome stability.
Assuntos
Replicação do DNA/genética , Ensaios de Triagem em Larga Escala , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Interpretação Estatística de Dados , Humanos , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Cyclin-dependent kinases 4 and 6 (CDK4/6) play a pivotal role in cell cycle and cancer development. Targeting CDK4/6 has demonstrated promising effects against breast cancer. However, resistance to CDK4/6 inhibitors (CDK4/6i), such as palbociclib, remains a substantial challenge in clinical settings. Using high-throughput combinatorial drug screening and genomic sequencing, we found that the microphthalmia-associated transcription factor (MITF) is activated via O-GlcNAcylation by O-GlcNAc transferase (OGT) in palbociclib-resistant breast cancer cells and tumors; O-GlcNAcylation of MITF at Serine 49 enhanced its interaction with importin α/ß, thus promoting its translocation to nuclei, where it suppressed palbociclib-induced senescence; inhibition of MITF or its O-GlcNAcylation re-sensitized resistant cells to palbociclib. Remarkably, clinical studies confirmed the activation of MITF in tumors from patients who are palbociclib-resistant or undergoing palbociclib treatment. Collectively, our studies shed light on a novel mechanism regulating palbociclib-resistance, and present clinical evidence for developing therapeutic approaches to treat CDK4/6i-resistant breast cancer patients.
RESUMO
Histone acetyltransferases play important roles in the regulation of chromatin structure and gene transcription. As one of the most important histone acetyltransferases, general control non-derepressible (Gcn) 5 has been linked to diverse cellular processes and tumorigenesis as well. We have recently identified a functional link between Gcn5 and acidic nucleoplasmic DNA-binding protein 1 (And-1) that is elevated in multiple cancer cells and is essential for Gcn5 protein stability. However, the mechanism by which And-1 regulates Gcn5 protein stability remains unknown. Here we show that the ablation of Cullin4-RING E3 ubiquitin ligase (CRL4) leads to the stabilization of Gcn5 in cells with depleted And-1, and Cdc10-dependent transcript 2 (Cdt2) serves as a substrate receptor protein of CRL4. Overexpression of Cdt2 reduces the Gcn5 protein levels, and CRL(Cdt2) is sufficient to ubiquitinate Gcn5 both in vivo and in vitro. And-1 stabilizes Gcn5 by impairing the interaction between Gcn5 and CRL(Cdt2) and thereby preventing Gcn5 ubiquitination and degradation. The degradation of Gcn5 is not dependent on proliferating cell nuclear antigen, an important player involved in CRL(Cdt2)-mediated protein degradation. Thus, CRL(Cdt2) and And-1 play an essential role in the regulation of Gcn5 protein stability. This study provides us with the mechanistic basis to develop alternative approaches to inhibit Gcn5 activity for cancer therapy.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Fatores de Transcrição de p300-CBP/metabolismo , Proteínas de Ligação a DNA/genética , Estabilidade Enzimática/fisiologia , Regulação da Expressão Gênica/fisiologia , Células HeLa , Humanos , Proteínas Nucleares/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina-Proteína Ligases/genética , Fatores de Transcrição de p300-CBP/genéticaRESUMO
OBJECTIVE: To evaluate the knowledge, attitudes and practices (KAP) of Chinese elite athletes about fluid replacement and urination. METHODS: A cross-section study was carried out among Chinese national and national youth teams from March to April 2020, using a pretested questionnaire. The 42-questions questionnaire was designed to assess the KAP regarding fluid replacement and urination. The questionnaire included knowledge of fluid replacement (KFR), attitudes of fluid replacement (AFR), knowledge of urination (KU), and attitudes of urination (AU), which were awarded 20 scoring points. Descriptive statistics, independent samples t-tests, one-way ANOVA, Pearson's correlation analysis, Multiple linear stepwise regression and Chi-square test were performed. RESULTS: A total of 779 valid questionnaires were collected and the effective rate is 98.4%. We finally conducted an assessment of 646 questionnaires of elite athletes. The mean score for KFR, AFR, KU, and AU was 2.8±1.3, 2.3±0.6, 3.0±1.5, and 2.1±0.8, respectively, with higher scores indicating positive hydration knowledge and attitudes. KFR and AFR scores of winter sports athletes were higher than those of summer sports athletes(P<0.05). Athletes who had lower athletic grades and training years had a worse KFR(P<0.05). Only 31.0% athletes knew that rehydration should be carried out before, during, and after training, which was scarcer among women, lower-athletic grades athletes, or athletes with lower training years (P<0.05). Male athletes had a worse KU but a better AU than female athletes(P<0.05). And athletes who were international-class athletic grades had the highest KU scores(P<0.05). The athletic grades and sport events were the main factors influencing the total scores of knowledge and attitudes (P<0.05, 95% CI -0.789--0.168,95% CI 0.025-1.040). Most of athletes tend to get hydration knowledge from internet. In practices, thirst is the main reason for rehydration (77.9%). The percentages of athletes with normal urine color (42.0%), frequency (75.0%,) and volume (20.0%) were low. CONCLUSIONS: These findings indicate that Chinese elite athletes did not have sufficient KAP on fluid replacement and urination, more marked in the individuals who were summer sport events, the lower athletic grades and in lower training years. It is recommended that education should be provided in the early stages of professional training for athletes.
Assuntos
Conhecimentos, Atitudes e Prática em Saúde , Micção , Adolescente , Atletas , China , Feminino , Humanos , Masculino , Inquéritos e QuestionáriosRESUMO
The Fanconi anemia (FA) pathway is essential for repairing DNA interstrand crosslinks (ICL). ICLs induce stalled DNA replication forks and trigger activation of the FA pathway by promoting recruitment of the FANCM/FAAP24/MHF complex to ICL sites. Given that stalled replication forks are proximal to ICL sites, fork-associated proteins may coordinate with FA factors to rapidly sense ICLs for activation of FA signaling. Here we report that And-1, a replisome protein, is critical for activation of the FA pathway by sensing ICL-stalled forks and recruiting the FANCM/FAAP24 complex to ICLs. In response to ICLs, And-1 rapidly accumulated at ICL-stalled forks in a manner dependent on ataxia telangiectasia and Rad3-related protein-induced phosphorylation at T826. And-1 phosphorylation triggered an intramolecular change that promoted the interaction of And-1 with FANCM/FAAP24, resulting in recruitment of the FANCM/FAAP24 complex to ICLs. Furthermore, p-T826 And-1 was elevated in cisplatin-resistant ovarian cancer cells, and activated And-1 contributed to cisplatin resistance. Collectively, these studies elucidate a mechanism by which And-1 regulates FA signaling and identify And-1 as a potential target for developing therapeutic approaches to treat platinum-resistant ovarian cancer. SIGNIFICANCE: This work shows that phosphorylation of And-1 by ATR activates Fanconi anemia signaling at interstrand crosslink-stalled replication forks by recruiting the FANCM/FAAP24 complex, revealing And-1 as a potential therapeutic target in cancer.
Assuntos
Anemia de Fanconi , Neoplasias Ovarianas , Cisplatino/farmacologia , DNA , Dano ao DNA , DNA Helicases/genética , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/genética , Anemia de Fanconi/tratamento farmacológico , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Feminino , HumanosRESUMO
Acidic nucleoplasmic DNA-binding protein 1 (And-1), an important factor for deoxyribonucleic acid (DNA) replication and repair, is overexpressed in many types of cancer but not in normal tissues. Although multiple independent studies have elucidated And-1 as a promising target gene for cancer therapy, an And-1 inhibitor has yet to be identified. Using an And-1 luciferase reporter assay to screen the Library of Pharmacologically Active Compounds (LOPAC) in a high throughput screening (HTS) platform, and then further screen the compound analog collection, we identified two potent And-1 inhibitors, bazedoxifene acetate (BZA) and an uncharacterized compound [(E)-5-(3,4-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol] (CH3), which specifically inhibit And-1 by promoting its degradation. Specifically, through direct interaction with And-1 WD40 domain, CH3 interrupts the polymerization of And-1. Depolymerization of And-1 promotes its interaction with E3 ligase Cullin 4B (CUL4B), resulting in its ubiquitination and subsequent degradation. Furthermore, CH3 suppresses the growth of a broad range of cancers. Moreover, And-1 inhibitors re-sensitize platinum-resistant ovarian cancer cells to platinum drugs in vitro and in vivo. Since BZA is an FDA approved drug, we expect a clinical trial of BZA-mediated cancer therapy in the near future. Taken together, our findings suggest that targeting And-1 by its inhibitors is a potential broad-spectrum anti-cancer chemotherapy regimen.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Neoplasias Ovarianas/tratamento farmacológico , Linhagem Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/uso terapêutico , Feminino , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/estatística & dados numéricos , Humanos , Neoplasias Ovarianas/fisiopatologiaRESUMO
The JAK2/STAT pathway is hyperactivated in many cancers, and such hyperactivation is associated with a poor clinical prognosis and drug resistance. The mechanism regulating JAK2 activity is complex. Although translocation of JAK2 between nucleus and cytoplasm is an important regulatory mechanism, how JAK2 translocation is regulated and what is the physiological function of this translocation remain largely unknown. Here, we found that protease SENP1 directly interacts with and deSUMOylates JAK2, and the deSUMOylation of JAK2 leads to its accumulation at cytoplasm, where JAK2 is activated. Significantly, this novel SENP1/JAK2 axis is activated in platinum-resistant ovarian cancer in a manner dependent on a transcription factor RUNX2 and activated RUNX2/SENP1/JAK2 is critical for platinum-resistance in ovarian cancer. To explore the application of anti-SENP1/JAK2 for treatment of platinum-resistant ovarian cancer, we found SENP1 deficiency or treatment by SENP1 inhibitor Momordin Ic significantly overcomes platinum-resistance of ovarian cancer. Thus, this study not only identifies a novel mechanism regulating JAK2 activity, but also provides with a potential approach to treat platinum-resistant ovarian cancer by targeting SENP1/JAK2 pathway.
Assuntos
Cisteína Endopeptidases/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Janus Quinase 2/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Platina/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Novel therapies are urgently needed for ovarian cancer, the deadliest gynecologic malignancy. Ovarian cancer has thus far been refractory to immunotherapies that stimulate the host immune system to recognize and kill cancer cells. This may be because of a suppressive tumor immune microenvironment and lack of recruitment and activation of immune cells that kill cancer cells. Our previous work showed that epigenetic drugs including DNA methyltransferase inhibitors and histone deacetylase 6 inhibitors (DNMTis and HDAC6is) individually increase immune signaling in cancer cells. We find that combining DNMTi and HDAC6i results in an amplified type I interferon response, leading to increased cytokine and chemokine expression and higher expression of the MHC I antigen presentation complex in human and mouse ovarian cancer cell lines. Treating mice bearing ID8 Trp53-/- ovarian cancer with HDAC6i/DNMTi led to an increase in tumor-killing cells such as IFNg+ CD8, NK, and NKT cells and a reversal of the immunosuppressive tumor microenvironment with a decrease in MDSCs and PD-1hi CD4 T cells, corresponding with an increase in survival. Thus combining the epigenetic modulators DNMTi and HDAC6i increases anti-tumor immune signaling from cancer cells and has beneficial effects on the ovarian tumor immune microenvironment.
Assuntos
DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Desacetilase 6 de Histona/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Feminino , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/citologia , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Taxa de SobrevidaRESUMO
The timely assembly of prereplicative complexes at replication origins is tightly controlled to ensure that genomic DNA is replicated once per cell cycle. The loss of geminin, a DNA replication inhibitor, causes rereplication that activates a G2/M checkpoint in human cancer cells. Fanconi anemia (FA) is an autosomal recessive and X-linked disorder associated with cancer susceptibility. Here we show that rereplication activates the FA pathway both for the activation of a G2/M checkpoint and for repair processes, like recruitment of RAD51. Both ATR and BRCA1 are required to activate the FA pathway. The G2/M checkpoint-mediated arrest of the cell cycle is critical for the prevention of both apoptosis and the accumulation of cells with rereplicated DNA, because the loss of ATR, BRCA1, or FANCA promotes apoptosis and suppresses the accumulation. The accumulation of cells with rereplicated DNA is restored by the artificial induction of a G2-phase arrest even when ATR, BRCA1, or FANCA is absent. Therefore, the ATR- and BRCA1-mediated FA pathway is required for the activation of a G2/M checkpoint and for DNA damage repair in response to the endogenous signal of rereplication. In its absence, the cells rapidly lose viability when faced with rereplication.