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1.
DNA Repair (Amst) ; 131: 103581, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37832251

RESUMEN

Cells possess an inherent and evolutionarily conserved ability to detect and respond to the presence of foreign and pathological 'self' nucleic acids. The result is the stimulation of innate immune responses, signalling to the host immune system that defence mechanisms are necessary to protect the organism. To date, there is a vast body of literature describing innate immune responses to various nucleic acid species, including dsDNA, ssDNA and ssRNA etc., however, there is limited information available on responses to R-loops. R-loops are 3-stranded nucleic acid structures that form during transcription, upon DNA damage and in various other settings. Emerging evidence suggests that innate immune responses may also exist for the detection of R-loop related nucleic acid structures, implicating R-loops as drivers of inflammatory states. In this review, we aim to summarise the evidence indicating that R-loops are immunogenic species that can trigger innate immune responses in physiological and pathological settings and discuss the implications of this in the study of various diseases and therapeutic development.


Asunto(s)
Neoplasias , Ácidos Nucleicos , Humanos , Estructuras R-Loop , Inmunidad Innata , ADN/genética , Neoplasias/patología , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo
2.
DNA Repair (Amst) ; 120: 103409, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36308822

RESUMEN

Genomic instability is a hallmark of tumourigenesis, influencing tumour development and progression. In particular, defects in the DNA damage response (DDR) have been extensively investigated and are known to shape therapeutic response. Since immune checkpoint blockade (ICB) therapy has been approved for treatment of tumours with defective mismatch repair the interplay between DDR pathway deficiency and the immune system has been of particular interest. The cGAS/STING signalling pathway has recently emerged as a key mediator of inflammation in response to DNA damage.This was identified through transcriptional profiling of BRCA1/2 deficient breast cancers and Fanconi Anaemia (FA) patient bone marrow, revealing a common transcriptional subgroup associated with BRCA1/2 and FA deficiency characterised by upregulation of innate immune signalling genes. Additionally, it is now apparent that the DNA damage arising from a multitude of DNA repair defects and DNA damage induced by some classical chemotherapies/radiation also has the ability to induce an innate immune response mediated by cGAS/STING activation. Here we review the role of intrinsic and extrinsic DNA damage in mediating immune activation and its context within tumourigenesis, as well as the potential therapeutic opportunities it represents for the treatment of cancer, such as combining DNA damaging agents with immunotherapies.


Asunto(s)
Anemia de Fanconi , Neoplasias , Humanos , Daño del ADN , Nucleotidiltransferasas/metabolismo , Reparación del ADN , Inestabilidad Genómica , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Carcinogénesis
3.
Cancer Res ; 82(5): 819-830, 2022 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-35027467

RESUMEN

Mutations in SF3B1 have been identified across several cancer types. This key spliceosome component promotes the efficient mRNA splicing of thousands of genes including those with crucial roles in the cellular response to DNA damage. Here, we demonstrate that depletion of SF3B1 specifically compromises homologous recombination (HR) and is epistatic with loss of BRCA1. More importantly, the most prevalent cancer-associated mutation in SF3B1, K700E, also affects HR efficiency and as a consequence, increases the cellular sensitivity to ionizing radiation and a variety of chemotherapeutic agents, including PARP inhibitors. In addition, the SF3B1 K700E mutation induced unscheduled R-loop formation, replication fork stalling, increased fork degradation, and defective replication fork restart. Taken together, these data suggest that tumor-associated mutations in SF3B1 induce a BRCA-like cellular phenotype that confers synthetic lethality to DNA-damaging agents and PARP inhibitors, which can be exploited therapeutically. SIGNIFICANCE: The cancer-associated SF3B1K700E mutation induces DNA damage via generation of genotoxic R-loops and stalled replication forks, defective homologous recombination, and increased replication fork degradation, which can be targeted with PARP inhibitors.


Asunto(s)
Neoplasias , Fosfoproteínas , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Factores de Empalme de ARN , Replicación del ADN , Humanos , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Fenotipo , Fosfoproteínas/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Factores de Empalme de ARN/genética , Mutaciones Letales Sintéticas
4.
Br J Cancer ; 126(2): 247-258, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34728791

RESUMEN

BACKGROUND: The DNA-damage immune-response (DDIR) signature is an immune-driven gene expression signature retrospectively validated as predicting response to anthracycline-based therapy. This feasibility study prospectively evaluates the use of this assay to predict neoadjuvant chemotherapy response in early breast cancer. METHODS: This feasibility study assessed the integration of a novel biomarker into clinical workflows. Tumour samples were collected from patients receiving standard of care neoadjuvant chemotherapy (FEC + /-taxane and anti-HER2 therapy as appropriate) at baseline, mid- and post-chemotherapy. Baseline DDIR signature scores were correlated with pathological treatment response. RNA sequencing was used to assess chemotherapy/response-related changes in biologically linked gene signatures. RESULTS: DDIR signature reports were available within 14 days for 97.8% of 46 patients (13 TNBC, 16 HER2 + ve, 27 ER + HER2-ve). Positive scores predicted response to treatment (odds ratio 4.67 for RCB 0-1 disease (95% CI 1.13-15.09, P = 0.032)). DDIR positivity correlated with immune infiltration and upregulated immune-checkpoint gene expression. CONCLUSIONS: This study validates the DDIR signature as predictive of response to neoadjuvant chemotherapy which can be integrated into clinical workflows, potentially identifying a subgroup with high sensitivity to anthracycline chemotherapy. Transcriptomic data suggest induction with anthracycline-containing regimens in immune restricted, "cold" tumours may be effective for immune priming. TRIAL REGISTRATION: Not applicable (non-interventional study). CRUK Internal Database Number 14232.


Asunto(s)
Neoplasias de la Mama/inmunología , Hidrocarburos Aromáticos con Puentes/uso terapéutico , Daño del ADN , Proteínas de la Membrana/metabolismo , Terapia Neoadyuvante/métodos , Recurrencia Local de Neoplasia/inmunología , Nucleotidiltransferasas/metabolismo , Taxoides/uso terapéutico , Adulto , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas de la Membrana/genética , Persona de Mediana Edad , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Nucleotidiltransferasas/genética , Resultado del Tratamiento
5.
Commun Biol ; 4(1): 484, 2021 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-33875784

RESUMEN

Genomic stability is critical for normal cellular function and its deregulation is a universal hallmark of cancer. Here we outline a previously undescribed role of COMMD4 in maintaining genomic stability, by regulation of chromatin remodelling at sites of DNA double-strand breaks. At break-sites, COMMD4 binds to and protects histone H2B from monoubiquitination by RNF20/RNF40. DNA damage-induced phosphorylation of the H2A-H2B heterodimer disrupts the dimer allowing COMMD4 to preferentially bind H2A. Displacement of COMMD4 from H2B allows RNF20/40 to monoubiquitinate H2B and for remodelling of the break-site. Consistent with this critical function, COMMD4-deficient cells show excessive elongation of remodelled chromatin and failure of both non-homologous-end-joining and homologous recombination. We present peptide-mapping and mutagenesis data for the potential molecular mechanisms governing COMMD4-mediated chromatin regulation at DNA double-strand breaks.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Biomarcadores de Tumor/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Histonas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Biomarcadores de Tumor/metabolismo , Células HEK293 , Células HeLa , Humanos
6.
NPJ Breast Cancer ; 7(1): 38, 2021 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-33824328

RESUMEN

Triple-negative breast cancer (TNBC) remains the most lethal breast cancer subtype with poor response rates to the current chemotherapies and a lack of additional effective treatment options. We have identified deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) as a critical gatekeeper that protects tumour DNA from the genotoxic misincorporation of uracil during treatment with standard chemotherapeutic agents commonly used in the FEC regimen. dUTPase catalyses the hydrolytic dephosphorylation of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP), providing dUMP for thymidylate synthase as part of the thymidylate biosynthesis pathway and maintaining low intracellular dUTP concentrations. This is crucial as DNA polymerase cannot distinguish between dUTP and deoxythymidylate triphosphate (dTTP), leading to dUTP misincorporation into DNA. Targeting dUTPase and inducing uracil misincorporation during the repair of DNA damage induced by fluoropyrimidines or anthracyclines represents an effective strategy to induce cell lethality. dUTPase inhibition significantly sensitised TNBC cell lines to fluoropyrimidines and anthracyclines through imbalanced nucleotide pools and increased DNA damage leading to decreased proliferation and increased cell death. These results suggest that repair of treatment-mediated DNA damage requires dUTPase to prevent uracil misincorporation and that inhibition of dUTPase is a promising strategy to enhance the efficacy of TNBC chemotherapy.

7.
Genes Chromosomes Cancer ; 60(5): 358-372, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33247475

RESUMEN

An underlying cause of breast cancers has been largely attributed to defects in the DNA damage response (DDR) pathway. In particular, the homologous recombination (HR) pathway repairs double-stranded breaks (DSBs) in DNA, ultimately protecting the cell from genomic instability and thus preventing the accumulation of transforming mutations. In line with this, mutations in a number of genes encoding HR proteins are a well-studied cause of HR deficiency (HRD), and, at the germline level, can confer risk to breast cancer but also occur somatically, contributing to sporadic breast cancer development, progression and response to therapy. Our understanding of the biological processes involved in HR and how these become compromised during breast cancer development has led to a better understanding of how HRD cells can be targeted with specific DNA damaging agents and/or with synthetic lethal targeting approaches such as PARP inhibition. Additionally, in vitro and preclinical modeling has supported the development of clinical trials to assess targeted therapies such as PARP inhibitors (PARPis), ultimately leading to development of therapies with greater clinical benefit. A number of challenges have been encountered, including resistance to therapy; however, addressing these challenges head-on and continually driving scientific research and clinical trials with innovative therapies will contribute to our ability to target HRD in breast cancers. Ongoing research efforts into HRD in breast cancer development are therefore essential, even in the era of targeted therapies, to provide innovative strategies for improved tumor responses.


Asunto(s)
Neoplasias de la Mama/genética , Reparación del ADN por Recombinación , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Femenino , Pruebas Genéticas/métodos , Mutación de Línea Germinal , Humanos , Terapia Molecular Dirigida/métodos
8.
J Transl Med ; 18(1): 339, 2020 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-32883299

RESUMEN

BACKGROUND: The cohesin complex plays a major role in folding the human genome into 3D structural domains. Mutations in members of the cohesin complex are known early drivers of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), with STAG2 the most frequently mutated complex member. METHODS: Here we use functional genomics (RNA-seq, ChIP-seq and HiChIP) to investigate the impact of chronic STAG2 loss on three-dimensional genome structure and transcriptional programming in a clinically relevant model of chronic STAG2 loss. RESULTS: The chronic loss of STAG2 led to loss of smaller loop domains and the maintenance/formation of large domains that, in turn, led to altered genome compartmentalisation. These changes in genome structure resulted in altered gene expression, including deregulation of the HOXA locus and the MAPK signalling pathway, resulting in increased sensitivity to MEK inhibition. CONCLUSIONS: The altered genomic architecture driven by the chronic loss of STAG2 results in altered gene expression that may contribute to leukaemogenesis and may be therapeutically targeted.


Asunto(s)
Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Proteínas de Ciclo Celular/genética , Cromatina/genética , Humanos , Leucemia Mieloide Aguda/genética , Mutación
9.
J Pathol Clin Res ; 6(2): 146-153, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32022473

RESUMEN

Multifocal (MF)/multicentric (MC) breast cancer is generally considered to be where two or more breast tumours are present within the same breast, and is seen in ~10% of breast cancer cases. This study investigates the prevalence of multifocality/multicentricity in a cohort of BRCA1/2 mutation carriers with breast cancer from Northern Ireland via cross-sectional analysis. Data from 211 women with BRCA1/2 mutations (BRCA1-91, BRCA2-120) and breast cancer were collected including age, tumour focality, size, type, grade and receptor profile. The prevalence of multifocality/multicentricity within this group was 25% but, within subgroups, prevalence amongst BRCA2 carriers was more than double that of BRCA1 carriers (p = 0.001). Women affected by MF/MC tumours had proportionately higher oestrogen receptor positivity (p = 0.001) and lower triple negativity (p = 0.004). These observations are likely to be driven by the higher BRCA2 mutation prevalence observed within this cohort. The odds of a BRCA2 carrier developing MF/MC cancer were almost four-fold higher than a BRCA1 carrier (odds ratio: 3.71, CI: 1.77-7.78, p = 0.001). These findings were subsequently validated in a second, large independent cohort of patients with BRCA-associated breast cancers from a UK-wide multicentre study. This confirmed a significantly higher prevalence of MF/MC tumours amongst BRCA2 mutation carriers compared with BRCA1 mutation carriers. This has important implications for clinicians involved in the treatment of BRCA2-associated breast cancer, both in the diagnostic process, in ensuring that tumour focality is adequately assessed to facilitate treatment decision-making, and for breast surgeons, particularly if breast conserving surgery is being considered as a treatment option for these patients.


Asunto(s)
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Mutación/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Mama/patología , Estudios de Cohortes , Estudios Transversales , Femenino , Predisposición Genética a la Enfermedad/genética , Humanos , Mastectomía Segmentaria
10.
Sci Rep ; 9(1): 2678, 2019 02 25.
Artículo en Inglés | MEDLINE | ID: mdl-30804405

RESUMEN

Myelodysplastic syndromes (MDS) are haematopoietic malignancies that are characterised by a heterogeneous clinical course. In recent years, sequencing efforts have uncovered recurrent somatic mutations within RNA splicing factors, including SF3B1, SRSF2, U2AF1 and ZRSR2. The most frequently mutated gene is SF3B1, mutated in 17% of MDS patients. While SF3B1 mutations and their effects on splicing have been well characterised, much remains to be explored about their more far-reaching effects on cellular homeostasis. Given that mRNA splicing and nuclear export are coordinated processes, we hypothesised that SF3B1 mutation might also affect export of certain mRNAs and that this may represent a targetable pathway for the treatment of SF3B1-mutant MDS. We used CRISPR/Cas9-genome editing to create isogenic cellular models. Comprehensive transcriptome and proteome profiling of these cells identified alterations in the splicing and export of components of the translational machinery, primarily tRNA synthetases, in response to the SF3B1 K700E mutation. While steady-state protein synthesis was unaffected, SF3B1 mutant cells were more sensitive to the clinically-relevant purine analogue, 8-azaguanine. In this study, we also demonstrated that 8-azaguanine affects splicing. Our results suggest that the simultaneous targeting of RNA metabolism and splicing by 8-azaguanine represents a therapeutic opportunity for SF3B1-mutant myelodysplastic syndromes.


Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Citoplasma/enzimología , Mutación , Síndromes Mielodisplásicos/genética , Fosfoproteínas/genética , Factores de Empalme de ARN/genética , Empalme del ARN , Aminoacil-ARNt Sintetasas/metabolismo , Línea Celular Tumoral , Edición Génica/métodos , Perfilación de la Expresión Génica/métodos , Células HEK293 , Humanos , Células K562 , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/terapia , Fosfoproteínas/metabolismo , Biosíntesis de Proteínas/genética , Proteoma/genética , Proteoma/metabolismo , Proteómica/métodos , Factores de Empalme de ARN/metabolismo
11.
Cancer Res ; 79(8): 2072-2075, 2019 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-30760519

RESUMEN

Modern methods of acquiring molecular data have improved rapidly in recent years, making it easier for researchers to collect large volumes of information. However, this has increased the challenge of recognizing interesting patterns within the data. Atlas Correlation Explorer (ACE) is a user-friendly workbench for seeking associations between attributes in The Cancer Genome Atlas (TCGA) database. It allows any combination of clinical and genomic data streams to be searched using an evolutionary algorithm approach. To showcase ACE, we assessed which RNA sequencing transcripts were associated with estrogen receptor (ESR1) in the TCGA breast cancer cohort. The analysis revealed already well-established associations with XBP1 and FOXA1, but also identified a strong association with CT62, a potential immunotherapeutic target with few previous associations with breast cancer. In conclusion, ACE can produce results for very large searches in a short time and will serve as an increasingly useful tool for biomarker discovery in the big data era. SIGNIFICANCE: ACE uses an evolutionary algorithm approach to perform large searches for associations between any combinations of data in the TCGA database.


Asunto(s)
Algoritmos , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Evolución Molecular , Genómica/métodos , Transcriptoma , Estudios de Cohortes , Femenino , Humanos , Programas Informáticos , Interfaz Usuario-Computador
12.
BMJ Open ; 8(12): e023115, 2018 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-30580266

RESUMEN

INTRODUCTION: BRCA1 mutation carriers have a significant lifetime risk of breast cancer, with their primary risk-reduction option being bilateral mastectomy. Preclinical work from our laboratory demonstrated that in BRCA1-deficient breast cells, oestrogen and its metabolites are capable of driving DNA damage and subsequent genomic instability, which are well-defined early events in BRCA1-related cancers. Based on this, we hypothesise that a chemopreventive approach which reduces circulating oestrogen levels may reduce DNA damage and genomic instability, thereby providing an alternative to risk-reducing surgery. METHODS AND ANALYSIS: 12 premenopausal women with pathogenic BRCA1 mutations and no previous risk-reducing surgery will be recruited from family history clinics. Participants will be allocated 1:1 to two arms. All will undergo baseline breast biopsies, blood and urine sampling, and quality of life questionnaires. Group A will receive goserelin 3.6 mg/28 days by subcutaneous injection, plus oral anastrozole 1 mg/day, for 12 weeks. Group B will receive oral tamoxifen 20 mg/day for 12 weeks. Following treatment, both groups will provide repeat biopsies, blood and urine samples, and questionnaires. Following a 1-month washout period, the groups will cross over, group A receiving tamoxifen and group B goserelin and anastrozole for a further 12 weeks. After treatment, biopsies, blood and urine samples, and questionnaires will be repeated. DNA damage will be assessed in core biopsies, while blood and urine samples will be used to measure oestrogen metabolite and DNA adduct levels. ETHICS AND DISSEMINATION: This study has ethical approval from the Office for Research Ethics Committees Northern Ireland (16/NI/0055) and the Medicines and Healthcare products Regulatory Agency (MHRA) (reference: 32485/0032/001-0001). The investigational medicinal products used in this trial are licensed and in common use, with well-documented safety information. Dissemination of results will be via high-impact journals and relevant national/international conferences. A copy of the results will be offered to the participants and be made available to patient support groups. TRIAL REGISTRATION NUMBER: EudraCT: 2016-001087-11; Pre-results.


Asunto(s)
Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Predisposición Genética a la Enfermedad/epidemiología , Aceptación de la Atención de Salud/estadística & datos numéricos , Ubiquitina-Proteína Ligasas/genética , Adulto , Anastrozol/uso terapéutico , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/prevención & control , Quimioprevención/métodos , Estudios Cruzados , Supervivencia sin Enfermedad , Estudios de Factibilidad , Femenino , Goserelina/uso terapéutico , Heterocigoto , Humanos , Mutación , Irlanda del Norte , Selección de Paciente , Premenopausia/fisiología , Pronóstico , Medición de Riesgo , Tasa de Supervivencia , Tamoxifeno/uso terapéutico , Resultado del Tratamiento
13.
JCO Precis Oncol ; 22018 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-30324181

RESUMEN

PURPOSE: Gene expression profiling can uncover biologic mechanisms underlying disease and is important in drug development. RNA sequencing (RNA-seq) is routinely used to assess gene expression, but costs remain high. Sample multiplexing reduces RNAseq costs; however, multiplexed samples have lower cDNA sequencing depth, which can hinder accurate differential gene expression detection. The impact of sequencing depth alteration on RNA-seq-based downstream analyses such as gene expression connectivity mapping is not known, where this method is used to identify potential therapeutic compounds for repurposing. METHODS: In this study, published RNA-seq profiles from patients with brain tumor (glioma) were assembled into two disease progression gene signature contrasts for astrocytoma. Available treatments for glioma have limited effectiveness, rendering this a disease of poor clinical outcome. Gene signatures were subsampled to simulate sequencing alterations and analyzed in connectivity mapping to investigate target compound robustness. RESULTS: Data loss to gene signatures led to the loss, gain, and consistent identification of significant connections. The most accurate gene signature contrast with consistent patient gene expression profiles was more resilient to data loss and identified robust target compounds. Target compounds lost included candidate compounds of potential clinical utility in glioma (eg, suramin, dasatinib). Lost connections may have been linked to low-abundance genes in the gene signature that closely characterized the disease phenotype. Consistently identified connections may have been related to highly expressed abundant genes that were ever-present in gene signatures, despite data reductions. Potential noise surrounding findings included false-positive connections that were gained as a result of gene signature modification with data loss. CONCLUSION: Findings highlight the necessity for gene signature accuracy for connectivity mapping, which should improve the clinical utility of future target compound discoveries.

14.
J Pathol ; 244(4): 445-459, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29520890

RESUMEN

Histological grading provides prognostic stratification of colorectal cancer (CRC) by scoring heterogeneous phenotypes. Features of aggressiveness include aberrant mitotic spindle configurations, chromosomal breakage, and bizarre multicellular morphology, but pathobiology is poorly understood. Protein kinase C zeta (PKCz) controls mitotic spindle dynamics, chromosome segregation, and multicellular patterns, but its role in CRC phenotype evolution remains unclear. Here, we show that PKCz couples genome segregation to multicellular morphology through control of interphase centrosome anchoring. PKCz regulates interdependent processes that control centrosome positioning. Among these, interaction between the cytoskeletal linker protein ezrin and its binding partner NHERF1 promotes the formation of a localized cue for anchoring interphase centrosomes to the cell cortex. Perturbation of these phenomena induced different outcomes in cells with single or extra centrosomes. Defective anchoring of a single centrosome promoted bipolar spindle misorientation, multi-lumen formation, and aberrant epithelial stratification. Collectively, these disturbances induce cribriform multicellular morphology that is typical of some categories of low-grade CRC. By contrast, defective anchoring of extra centrosomes promoted multipolar spindle formation, chromosomal instability (CIN), disruption of glandular morphology, and cell outgrowth across the extracellular matrix interface characteristic of aggressive, high-grade CRC. Because PKCz enhances apical NHERF1 intensity in 3D epithelial cultures, we used an immunohistochemical (IHC) assay of apical NHERF1 intensity as an indirect readout of PKCz activity in translational studies. We show that apical NHERF1 IHC intensity is inversely associated with multipolar spindle frequency and high-grade morphology in formalin-fixed human CRC samples. To conclude, defective PKCz control of interphase centrosome anchoring may underlie distinct categories of mitotic slippage that shape the development of low- or high-grade CRC phenotypes. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Asunto(s)
Centrosoma/enzimología , Neoplasias Colorrectales/enzimología , Interfase , Proteína Quinasa C/metabolismo , Células CACO-2 , Proliferación Celular , Forma de la Célula , Inestabilidad Cromosómica , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Humanos , Clasificación del Tumor , Fenotipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteína Quinasa C/genética , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo
15.
Nucleic Acids Res ; 45(22): 12816-12833, 2017 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-29112714

RESUMEN

mRNA splicing and export plays a key role in the regulation of gene expression, with recent evidence suggesting an additional layer of regulation of gene expression and cellular function through the selective splicing and export of genes within specific pathways. Here we describe a role for the RNA processing factors THRAP3 and BCLAF1 in the regulation of the cellular DNA damage response (DDR) pathway, a key pathway involved in the maintenance of genomic stability and the prevention of oncogenic transformation. We show that loss of THRAP3 and/or BCLAF1 leads to sensitivity to DNA damaging agents, defective DNA repair and genomic instability. Additionally, we demonstrate that this phenotype can be at least partially explained by the role of THRAP3 and BCLAF1 in the selective mRNA splicing and export of transcripts encoding key DDR proteins, including the ATM kinase. Moreover, we show that cancer associated mutations within THRAP3 result in deregulated processing of THRAP3/BCLAF1-regulated transcripts and consequently defective DNA repair. Taken together, these results suggest that THRAP3 and BCLAF1 mutant tumors may be promising targets for DNA damaging chemotherapy.


Asunto(s)
Transporte Activo de Núcleo Celular/genética , Daño del ADN , Proteínas de Unión al ADN/genética , Empalme del ARN , Proteínas Represoras/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica/métodos , Células HEK293 , Humanos , Hibridación Fluorescente in Situ , Microscopía Fluorescente , Mutación , Interferencia de ARN , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo
16.
J Natl Cancer Inst ; 109(1)2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27707838

RESUMEN

Background: Previously we identified a DNA damage response-deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance. Methods: We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided. Results: We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher's exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response-proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response-proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle-specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner. Conclusions: We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint-based therapies.


Asunto(s)
Neoplasias de la Mama/inmunología , Daño del ADN/inmunología , ADN/análisis , Inmunidad Innata , Leucocitos Mononucleares/fisiología , Linfocitos Infiltrantes de Tumor , Proteínas de la Membrana/metabolismo , Antígeno B7-H1/metabolismo , Neoplasias de la Mama/genética , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Línea Celular Tumoral , Quimiocina CCL5/metabolismo , Quimiocina CXCL10/metabolismo , Quimiotaxis/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Citosol/química , Femenino , Humanos , Inmunohistoquímica , Factor 3 Regulador del Interferón/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Fase S/genética , Transducción de Señal
17.
Wiley Interdiscip Rev RNA ; 7(5): 604-19, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27126972

RESUMEN

Despite consistent research into the molecular principles of the DNA damage repair pathway for almost two decades, it has only recently been found that RNA metabolism is very tightly related to this pathway, and the two ancient biochemical mechanisms act in alliance to maintain cellular genomic integrity. The close links between these pathways are well exemplified by examining the base excision repair pathway, which is now well known for dual roles of many of its members in DNA repair and RNA surveillance, including APE1, SMUG1, and PARP1. With additional links between these pathways steadily emerging, this review aims to provide a summary of the emerging roles for DNA repair proteins in the post-transcriptional regulation of RNAs. WIREs RNA 2016, 7:604-619. doi: 10.1002/wrna.1353 For further resources related to this article, please visit the WIREs website.


Asunto(s)
Enzimas Reparadoras del ADN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo
18.
Cancer Res ; 75(11): 2159-65, 2015 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-25870146

RESUMEN

Ataxia telangiectasia mutated (ATM) is an important signaling molecule in the DNA damage response (DDR). ATM loss of function can produce a synthetic lethal phenotype in combination with tumor-associated mutations in FA/BRCA pathway components. In this study, we took an siRNA screening strategy to identify other tumor suppressors that, when inhibited, similarly sensitized cells to ATM inhibition. In this manner, we determined that PTEN and ATM were synthetically lethal when jointly inhibited. PTEN-deficient cells exhibited elevated levels of reactive oxygen species, increased endogenous DNA damage, and constitutive ATM activation. ATM inhibition caused catastrophic DNA damage, mitotic cell cycle arrest, and apoptosis specifically in PTEN-deficient cells in comparison with wild-type cells. Antioxidants abrogated the increase in DNA damage and ATM activation in PTEN-deficient cells, suggesting a requirement for oxidative DNA damage in the mechanism of cell death. Lastly, the ATM inhibitor KU-60019 was specifically toxic to PTEN mutant cancer cells in tumor xenografts and reversible by reintroduction of wild-type PTEN. Together, our results offer a mechanistic rationale for clinical evaluation of ATM inhibitors in PTEN-deficient tumors.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Neoplasias Colorrectales/genética , Daño del ADN/efectos de los fármacos , Fosfohidrolasa PTEN/genética , Animales , Apoptosis/efectos de los fármacos , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Reparación del ADN/efectos de los fármacos , Células HCT116 , Xenoinjertos , Humanos , Ratones , Mitosis/efectos de los fármacos , Morfolinas/administración & dosificación , Fosfohidrolasa PTEN/biosíntesis , ARN Interferente Pequeño , Tioxantenos/administración & dosificación
19.
Cell Rep ; 11(1): 149-63, 2015 Apr 07.
Artículo en Inglés | MEDLINE | ID: mdl-25818296

RESUMEN

Cells experience damage from exogenous and endogenous sources that endanger genome stability. Several cellular pathways have evolved to detect DNA damage and mediate its repair. Although many proteins have been implicated in these processes, only recent studies have revealed how they operate in the context of high-ordered chromatin structure. Here, we identify the nuclear oncogene SET (I2PP2A) as a modulator of DNA damage response (DDR) and repair in chromatin surrounding double-strand breaks (DSBs). We demonstrate that depletion of SET increases DDR and survival in the presence of radiomimetic drugs, while overexpression of SET impairs DDR and homologous recombination (HR)-mediated DNA repair. SET interacts with the Kruppel-associated box (KRAB)-associated co-repressor KAP1, and its overexpression results in the sustained retention of KAP1 and Heterochromatin protein 1 (HP1) on chromatin. Our results are consistent with a model in which SET-mediated chromatin compaction triggers an inhibition of DNA end resection and HR.


Asunto(s)
Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Chaperonas de Histonas/genética , Reparación del ADN por Recombinación/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/biosíntesis , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN/genética , Proteínas de Unión al ADN/genética , Heterocromatina/genética , Chaperonas de Histonas/antagonistas & inhibidores , Chaperonas de Histonas/metabolismo , Humanos , Proteínas Represoras/biosíntesis , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Proteína 28 que Contiene Motivos Tripartito
20.
FEBS J ; 282(4): 630-46, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25400280

RESUMEN

BRCA1 is a major breast and ovarian cancer susceptibility gene, with mutations in this gene predisposing women to a very high risk of developing breast and ovarian tumours. BRCA1 primarily functions to maintain genomic stability via critical roles in DNA repair, cell cycle checkpoint control, transcriptional regulation, apoptosis and mRNA splicing. As a result, BRCA1 mutations often result in defective DNA repair, genomic instability and sensitivity to DNA damaging agents. BRCA1 carries out these different functions through its ability to interact, and form complexes with, a vast array of proteins involved in multiple cellular processes, all of which are considered to contribute to its function as a tumour suppressor. This review discusses and highlights recent research into the functions of BRCA1-related protein complexes and their roles in maintaining genomic stability and tumour suppression.


Asunto(s)
Proteína BRCA1/metabolismo , Inestabilidad Genómica/fisiología , Animales , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Daño del ADN/genética , Daño del ADN/fisiología , Reparación del ADN/genética , Reparación del ADN/fisiología , Femenino , Inestabilidad Genómica/genética , Humanos
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