RESUMEN
Zhao et al. (2022) demonstrate that HIV Tat-specific factor 1, an RPA PARylation reader, recruits Topoisomerase IIß-binding protein 1 to double-strand break sites specifically in the S phase of the cell cycle to promote homologous recombination.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , ADN-Topoisomerasas de Tipo II/genética , Recombinación Homóloga , Poli ADP Ribosilación , Fase SRESUMEN
53BP1 influences genome stability via two independent mechanisms: (1) regulating DNA double-strand break (DSB) repair and (2) enhancing p53 activity. We discovered a protein, Tudor-interacting repair regulator (TIRR), that associates with the 53BP1 Tudor domain and prevents its recruitment to DSBs. Here, we elucidate how TIRR affects 53BP1 function beyond its recruitment to DSBs and biochemically links the two distinct roles of 53BP1. Loss of TIRR causes an aberrant increase in the gene transactivation function of p53, affecting several p53-mediated cell-fate programs. TIRR inhibits the complex formation between the Tudor domain of 53BP1 and a dimethylated form of p53 (K382me2) that is poised for transcriptional activation of its target genes. TIRR mRNA expression levels negatively correlate with the expression of key p53 target genes in breast and prostate cancers. Further, TIRR loss is selectively not tolerated in p53-proficient tumors. Therefore, we establish that TIRR is an important inhibitor of the 53BP1-p53 complex.
Asunto(s)
Linaje de la Célula/genética , Proteínas de Unión al ARN/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Sitios de Unión , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Linaje de la Célula/fisiología , ADN/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Histonas/metabolismo , Humanos , Unión Proteica , Proteínas de Unión al ARN/fisiología , Dominio Tudor , Proteína p53 Supresora de Tumor/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/fisiologíaRESUMEN
While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A/PABIR1 confers cellular resistance to CHK1 inhibitors (CHK1is) and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase that dephosphorylates the WEE1 protein and rescues WEE1 from ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1is. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1i plus a WEE1 inhibitor can overcome CHK1i resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1i sensitivity or resistance.
Asunto(s)
Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas/metabolismo , Pirazinas/farmacología , Pirazoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Daño del ADN/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Nucleares/metabolismo , Fosfoproteínas/fisiología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Tirosina Quinasas/genética , Pirazinas/metabolismo , Pirazoles/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
During the past decade, evolutionarily conserved microRNAs (miRNAs) have been characterized as regulators of almost every cellular process and signalling pathway. There is now emerging evidence that this new class of regulators also impinges on the DNA damage response (DDR). Both miRNAs and other small non-coding RNAs (ncRNAs) are induced at DNA breaks and mediate the repair process. These intriguing observations raise the possibility that crosstalk between ncRNAs and the DDR might provide a means of efficient and accurate DNA repair and facilitate the maintenance of genomic stability.
Asunto(s)
Roturas del ADN , Reparación del ADN , MicroARNs/genética , MicroARNs/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismo , Ciclo Celular/genética , Fenómenos Fisiológicos Celulares , Transducción de SeñalRESUMEN
The protection of telomere ends by the shelterin complex prevents DNA damage signalling and promiscuous repair at chromosome ends. Evidence suggests that the 3' single-stranded telomere end can assemble into a lasso-like t-loop configuration1,2, which has been proposed to safeguard chromosome ends from being recognized as DNA double-strand breaks2. Mechanisms must also exist to transiently disassemble t-loops to allow accurate telomere replication and to permit telomerase access to the 3' end to solve the end-replication problem. However, the regulation and physiological importance of t-loops in the protection of telomere ends remains unknown. Here we identify a CDK phosphorylation site in the shelterin subunit at Ser365 of TRF2, whose dephosphorylation in S phase by the PP6R3 phosphatase provides a narrow window during which the RTEL1 helicase can transiently access and unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 at Ser365 outside of S phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate activation of ATM, but also counteracts replication conflicts at DNA secondary structures that arise within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates the assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response.
Asunto(s)
Ciclo Celular , Quinasas Ciclina-Dependientes/metabolismo , Fosfoserina/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/química , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , ADN/biosíntesis , ADN/química , ADN/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN , ADN Helicasas/metabolismo , Reparación del ADN , Replicación del ADN , Fibroblastos , Genoma/genética , Células HEK293 , Humanos , Ratones , Mutación , Fenotipo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Fase S , Complejo Shelterina , Telomerasa/metabolismo , Telómero/genética , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
The 53BP1-RIF1 pathway restricts the resection of DNA double-strand breaks (DSBs) and promotes blunt end-ligation by non-homologous end joining (NHEJ) repair. The Shieldin complex is a downstream effector of the 53BP1-RIF1 pathway. Here, we identify a component of this pathway, CCAR2/DBC1, which is also required for restriction of DNA end-resection. CCAR2 co-immunoprecipitates with the Shieldin complex, and knockout of CCAR2 in a BRCA1-deficient cell line results in elevated DSB end-resection, RAD51 loading, and PARP inhibitor (PARPi) resistance. Knockout of CCAR2 is epistatic with knockout of other Shieldin proteins. The S1-like RNA-binding domain of CCAR2 is required for its interaction with the Shieldin complex and for suppression of DSB end-resection. CCAR2 functions downstream of the Shieldin complex, and CCAR2 knockout cells have delayed resolution of Shieldin complex foci. Forkhead-associated (FHA)-dependent targeting of CCAR2 to DSB sites re-sensitized BRCA1-/-SHLD2-/- cells to PARPi. Taken together, CCAR2 is a functional component of the 53BP1-RIF1 pathway, promotes the refill of resected DSBs, and suppresses homologous recombination.
Asunto(s)
Roturas del ADN de Doble Cadena , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Reparación del ADN por Unión de Extremidades , Recombinación Homóloga , ADNRESUMEN
OBJECTIVE: To determine whether a multimodal assay combining serum microRNA with protein biomarkers and metadata improves triage assessment of an adnexal mass. METHODS: Serum samples from 468 training subjects (191 cancer cases and 277 benign adnexal mass controls or healthy controls) were analyzed for seven protein biomarkers and 180 miRNA. Circulating analyte data were combined with age and menopausal status (metadata) into a neural network model to classify samples as cases or controls. Forward regression with ten-fold cross-validation minimized the dimensionality of the model while maximizing linear separation between cases and controls. Model validation proceeded using both internal (44 cases and 56 controls) and external validation sets (51 cases and 59 controls). RESULTS: The total study population comprised 678 subjects, including 286 cases and 392 controls. Overall, 290 (43%) of the subjects were premenopausal. A panel of 10 miRNA delivered optimal performance when combined with protein and metadata features. The combined model improved the Receiver Operator Characteristic Area Under the Curve (ROC AUC) on the internal (AUC = 0.9; 95% CI 0.81-0.95) and external validation sets (AUC = 0.95; 95% CI 0.90-0.98) compared to miRNA alone or proteins plus metadata (without miRNA). On external validation, the combined model offered 92% sensitivity at 80% specificity overall, with 80% and 100% sensitivity for early and late-stage cancers, respectively, including 78% sensitivity for early-stage, serous ovarian cancers and 82% sensitivity for early-stage, non-serous cancers. CONCLUSIONS: A multimodal assay combining miRNA with protein biomarkers, age, and menopausal status improves surgical triage of an adnexal mass.
RESUMEN
Limited DNA end resection is the key to impaired homologous recombination in BRCA1-mutant cancer cells. Here, using a loss-of-function CRISPR screen, we identify DYNLL1 as an inhibitor of DNA end resection. The loss of DYNLL1 enables DNA end resection and restores homologous recombination in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and inhibitors of poly(ADP-ribose) polymerase. Low BRCA1 expression correlates with increased chromosomal aberrations in primary ovarian carcinomas, and the junction sequences of somatic structural variants indicate diminished homologous recombination. Concurrent decreases in DYNLL1 expression in carcinomas with low BRCA1 expression reduced genomic alterations and increased homology at lesions. In cells, DYNLL1 limits nucleolytic degradation of DNA ends by associating with the DNA end-resection machinery (MRN complex, BLM helicase and DNA2 endonuclease). In vitro, DYNLL1 binds directly to MRE11 to limit its end-resection activity. Therefore, we infer that DYNLL1 is an important anti-resection factor that influences genomic stability and responses to DNA-damaging chemotherapy.
Asunto(s)
Proteína BRCA1/deficiencia , Dineínas Citoplasmáticas/metabolismo , ADN/metabolismo , Genes BRCA1 , Proteína Homóloga de MRE11/metabolismo , Reparación del ADN por Recombinación , Proteína BRCA1/genética , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Aberraciones Cromosómicas , Daño del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Edición Génica , Inestabilidad Genómica/efectos de los fármacos , Recombinación Homóloga/efectos de los fármacos , Humanos , Mutación , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Platino (Metal)/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Unión Proteica , Reparación del ADN por Recombinación/efectos de los fármacos , Factores de Transcripción/metabolismoRESUMEN
P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, Tudor interacting repair regulator (TIRR), that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, the protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates 53BP1 and recruits RAP1-interacting factor 1 (RIF1) to dissociate the 53BP1-TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to double-strand breaks. Depletion of TIRR destabilizes 53BP1 in the nuclear-soluble fraction and alters the double-strand break-induced protein complex centring 53BP1. These findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1.
Asunto(s)
Proteínas Portadoras/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/antagonistas & inhibidores , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Sitios de Unión , Roturas del ADN de Doble Cadena , Reparación del ADN , Femenino , Humanos , Metilación , Ratones , Ratones Endogámicos C57BL , Fosforilación , Unión Proteica , Dominios Proteicos , Proteínas de Unión al ARN , Proteínas de Unión a Telómeros/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/químicaRESUMEN
Formation of G-quadruplex (G4) DNA structures in key regulatory regions in the genome has emerged as a secondary structure-based epigenetic mechanism for regulating multiple biological processes including transcription, replication, and telomere maintenance. G4 formation (folding), stabilization, and unfolding must be regulated to coordinate G4-mediated biological functions; however, how cells regulate the spatiotemporal formation of G4 structures in the genome is largely unknown. Here, we demonstrate that endogenous oxidized guanine bases in G4 sequences and the subsequent activation of the base excision repair (BER) pathway drive the spatiotemporal formation of G4 structures in the genome. Genome-wide mapping of occurrence of Apurinic/apyrimidinic (AP) site damage, binding of BER proteins, and G4 structures revealed that oxidized base-derived AP site damage and binding of OGG1 and APE1 are predominant in G4 sequences. Loss of APE1 abrogated G4 structure formation in cells, which suggests an essential role of APE1 in regulating the formation of G4 structures in the genome. Binding of APE1 to G4 sequences promotes G4 folding, and acetylation of APE1, which enhances its residence time, stabilizes G4 structures in cells. APE1 subsequently facilitates transcription factor loading to the promoter, providing mechanistic insight into the role of APE1 in G4-mediated gene expression. Our study unravels a role of endogenous oxidized DNA bases and APE1 in controlling the formation of higher-order DNA secondary structures to regulate transcription beyond its well-established role in safeguarding the genomic integrity.
Asunto(s)
Daño del ADN , Reparación del ADN/fisiología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , G-Cuádruplex , Células A549 , Acetilación , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Expresión Génica , Genes myc , Genoma Humano , Guanina/química , Guanina/metabolismo , Células HCT116 , Humanos , Oxidación-Reducción , Estrés Oxidativo/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas p21(ras)/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
PURPOSE: PARP inhibitor resistance may be overcome by combinatorial strategies with agents that disrupt homologous recombination repair (HRR). Multiple HRR pathway components are HSP90 clients, so that HSP90 inhibition leads to abrogation of HRR and sensitisation to PARP inhibition. We performed in vivo preclinical studies of the HSP90 inhibitor onalespib with olaparib and conducted a Phase 1 combination study. PATIENTS AND METHODS: Tolerability and efficacy studies were performed in patient-derived xenograft(PDX) models of ovarian cancer. Clinical safety, tolerability, steady-state pharmacokinetics and preliminary efficacy of olaparib and onalespib were evaluated using a standard 3 + 3 dose-escalation design. RESULTS: Olaparib/onalespib exhibited anti-tumour activity against BRCA1-mutated PDX models with acquired PARPi resistance and PDX models with RB-pathway alterations(CDKN2A loss and CCNE1 overexpression). Phase 1 evaluation revealed that dose levels up to olaparib 300 mg/onalespib 40 mg and olaparib 200 mg/onalespib 80 mg were safe without dose-limiting toxicities. Coadministration of olaparib and onalespib did not appear to affect the steady-state pharmacokinetics of either agent. There were no objective responses, but disease stabilisation ≥24 weeks was observed in 7/22 (32%) evaluable patients including patients with BRCA-mutated ovarian cancers and acquired PARPi resistance and patients with tumours harbouring RB-pathway alterations. CONCLUSIONS: Combining onalespib and olaparib was feasible and demonstrated preliminary evidence of anti-tumour activity.
Asunto(s)
Antineoplásicos , Neoplasias Ováricas , Antineoplásicos/uso terapéutico , Carcinoma Epitelial de Ovario , Proteínas HSP90 de Choque Térmico , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Ftalazinas/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéuticoRESUMEN
Excluding 53BP1 from chromatin is required to attenuate the DNA damage response during mitosis, yet the functional relevance and regulation of this exclusion are unclear. Here we show that 53BP1 is phosphorylated during mitosis on two residues, T1609 and S1618, located in its well-conserved ubiquitination-dependent recruitment (UDR) motif. Phosphorylating these sites blocks the interaction of the UDR motif with mononuclesomes containing ubiquitinated histone H2A and impedes binding of 53BP1 to mitotic chromatin. Ectopic recruitment of 53BP1-T1609A/S1618A to mitotic DNA lesions was associated with significant mitotic defects that could be reversed by inhibiting nonhomologous end-joining. We also reveal that protein phosphatase complex PP4C/R3ß dephosphorylates T1609 and S1618 to allow the recruitment of 53BP1 to chromatin in G1 phase. Our results identify key sites of 53BP1 phosphorylation during mitosis, identify the counteracting phosphatase complex that restores the potential for DDR during interphase, and establish the physiological importance of this regulation.
Asunto(s)
Roturas del ADN de Doble Cadena , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Fase G1 , Células HeLa , Humanos , Mitosis , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Unión Proteica , Transporte de Proteínas , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
OBJECTIVE: Uterine serous carcinoma (USC) is an aggressive subtype of endometrial cancer associated with worse survival outcomes in African American (AA) patients. This study evaluated tumor miRNA expression by race, clinical and tumor characteristics, and survival outcomes. METHODS: FFPE tumor tissue from hysterectomy specimens was identified for 29 AA cases. Case matching was performed by computer-based random assignment in a 1:1 ratio with Caucasian controls based on age, stage and histologic subtype (pure vs. mixed). RNA was extracted from 77 specimens (54 tumors and 23 matched normal endometrium). MicroRNA array profiling was performed by microRNA Hi-Power Labeling (Hy3/Hy5) and hybridization to miRCURY LNA microRNA Array 7th Gen. RESULTS: Clinical and treatment characteristics were similar for cases and controls, although use of adjuvant radiation was less common in African Americans (p = 0.03). Of 968 miRNAs analyzed, 649 were differentially expressed in normal endometrium vs. tumor. When compared by race, histologic subtype, stage or presence of LVI, no differentially expressed miRNAs were identified. In patients with disease recurrence at 3 years, the three most upregulated miRNAs were miR-1, miR-21-5p and miR-223. Of these, increased miR-223 expression (>median) was associated with worse OS (p = 0.0496) in an independent dataset (TCGA dataset) comprising of 140 patients with USC (mixed or pure serous). After adjusting for age, ethnicity and BMI, upregulation of miR-223 remained risk factor for death (adjusted HR 2.87, 95% CI 1.00-8.27). CONCLUSIONS: MiRNA profiling did not identify biological differences between AA and Caucasian patients with USC. Upregulation of miR-223 may be a prognostic factor in USC.
Asunto(s)
Negro o Afroamericano/genética , Cistadenocarcinoma Seroso/genética , MicroARNs/genética , Neoplasias Uterinas/genética , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Estudios de Cohortes , Cistadenocarcinoma Seroso/etnología , Cistadenocarcinoma Seroso/patología , Cistadenocarcinoma Seroso/terapia , Femenino , Perfilación de la Expresión Génica , Disparidades en el Estado de Salud , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Regulación hacia Arriba , Neoplasias Uterinas/etnología , Neoplasias Uterinas/patología , Neoplasias Uterinas/terapiaRESUMEN
BACKGROUND: High-grade serous ovarian cancers show increased replication stress, rendering cells vulnerable to ATR inhibition because of near universal loss of the G1/S checkpoint (through deleterious TP53 mutations), premature S phase entry (due to CCNE1 amplification, RB1 loss, or CDKN2A mRNA downregulation), alterations of homologous recombination repair genes, and expression of oncogenic drivers (through MYC amplification and other mechanisms). We hypothesised that the combination of the selective ATR inhibitor, berzosertib, and gemcitabine could show acceptable toxicity and superior efficacy to gemcitabine alone in high-grade serous ovarian cancer. METHODS: In this multicentre, open-label, randomised, phase 2 study, 11 different centres in the US Experimental Therapeutics Clinical Trials Network enrolled women (aged ≥18 years) with recurrent, platinum-resistant high-grade serous ovarian cancer (determined histologically) and Eastern Cooperative Oncology Group performance status of 0 or 1, who had unlimited previous lines of cytotoxic therapy in the platinum-sensitive setting but no more than one line of cytotoxic therapy in the platinum-resistant setting. Eligible patients were randomly assigned (1:1) to receive intravenous gemcitabine (1000 mg/m2) on day 1 and day 8, or gemcitabine plus intravenous berzosertib (210 mg/m2) on day 2 and day 9 of a 21-day cycle until disease progression or intolerable toxicity. Randomisation was done centrally using the Theradex Interactive Web Response System, stratified by platinum-free interval, and with a permuted block size of six. Following central randomisation, patients and investigators were not masked to treatment assignment. The primary endpoint was investigator-assessed progression-free survival, and analyses included all patients who received at least one dose of the study drugs. The study is registered with ClinicalTrials.gov, NCT02595892, and is active but closed to enrolment. FINDINGS: Between Feb 14, 2017, and Sept 7, 2018, 88 patients were assessed for eligibility, of whom 70 were randomly assigned to treatment with gemcitabine alone (36 patients) or gemcitabine plus berzosertib (34 patients). At the data cutoff date (Feb 21, 2020), the median follow-up was 53·2 weeks (25·6-81·8) in the gemcitabine plus berzosertib group and 43·0 weeks (IQR 23·2-69·1) in the gemcitabine alone group. Median progression-free survival was 22·9 weeks (17·9-72·0) for gemcitabine plus berzosertib and 14·7 weeks (90% CI 9·7-36·7) for gemcitabine alone (hazard ratio 0·57, 90% CI 0·33-0·98; one-sided log-rank test p=0·044). The most common treatment-related grade 3 or 4 adverse events were decreased neutrophil count (14 [39%] of 36 patients in the gemcitabine alone group vs 16 [47%] of 34 patients in the gemcitabine plus berzosertib group) and decreased platelet count (two [6%] vs eight [24%]). Serious adverse events were observed in ten (28%) patients in the gemcitabine alone group and nine (26%) patients in the gemcitabine plus berzosertib group. There was one treatment-related death in the gemcitabine alone group due to sepsis and one treatment-related death in the gemcitabine plus berzosertib group due to pneumonitis. INTERPRETATION: To our knowledge, this is the first randomised study of an ATR inhibitor in any tumour type. This study shows a benefit of adding berzosertib to gemcitabine in platinum-resistant high-grade serous ovarian cancer. This combination warrants further investigation in this setting. FUNDING: US National Cancer Institute.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Cistadenocarcinoma Seroso/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Ováricas/tratamiento farmacológico , Terapia Recuperativa , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Cistadenocarcinoma Seroso/patología , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Femenino , Estudios de Seguimiento , Humanos , Isoxazoles/administración & dosificación , Persona de Mediana Edad , Clasificación del Tumor , Neoplasias Ováricas/patología , Platino (Metal)/farmacología , Pirazinas/administración & dosificación , Tasa de Supervivencia , Adulto Joven , GemcitabinaRESUMEN
DNA repair genes are commonly altered in metastatic prostate cancer, but BRCA1 mutations are rare. Preliminary studies suggest that higher tumor expression of the BRCA1 protein may be associated with worse prognosis. We undertook a prospective study among men with prostate cancer in the Health Professionals Follow-up Study and evaluated BRCA1 via immunohistochemical staining on tissue microarrays. BRCA1 was expressed in 60 of 589 tumors. Prevalence of BRCA1 positivity was 43% in the 14 men with metastases at diagnosis compared with 9% in non-metastatic tumors [difference, 33 percentage points; 95% confidence interval (CI), 7-59]. BRCA1-positive tumors had 2.16-fold higher Ki-67 proliferative indices (95% CI, 1.18-3.95), higher tumor aneuploidy as predicted from whole-transcriptome profiling, and higher Gleason scores. Among the 575 patients with non-metastatic disease at diagnosis, we evaluated the association between BRCA1 expression and development of lethal disease (metastasis or cancer-specific death, 69 events) during long-term follow-up (median, 18.3 years). A potential weak association of BRCA1 positivity with lethal disease (hazard ratio, 1.61; 95% CI, 0.82-3.15) was attenuated when adjusting for age, Gleason score and clinical stage (hazard ratio, 1.11; 95% CI, 0.54-2.29). In summary, BRCA1 protein expression is a feature of more proliferative and more aneuploid prostate tumors and is more common in metastatic disease. While not well suited as a prognostic biomarker in primary prostate cancer, BRCA1 protein expression may be most relevant in advanced disease.
Asunto(s)
Proteína BRCA1/genética , Reparación del ADN/genética , Neoplasias de Tejido Óseo/genética , Neoplasias de la Próstata/genética , Adulto , Anciano , Biomarcadores de Tumor , Progresión de la Enfermedad , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Clasificación del Tumor , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Neoplasias de Tejido Óseo/patología , Neoplasias de Tejido Óseo/secundario , Neoplasias de la Próstata/patologíaRESUMEN
BACKGROUND: The consensus on how to choose a reference gene for serum or plasma miRNA expression qPCR studies has not been reached and none of the potential candidates have yet been convincingly validated. We proposed a new in silico approach of finding a suitable reference for human, circulating miRNAs and identified a new set of endogenous reference miRNA based on miRNA profiling experiments from Gene Expression Omnibus. We used 3 known normalization algorithms (NormFinder, BestKeeper, GeNorm) to calculate a new normalization score. We searched for a universal set of endogenous miRNAs and validated our findings on 2 new datasets using our approach. RESULTS: We discovered and validated a set of 13 miRNAs (miR-222, miR-92a, miR-27a, miR-17, miR-24, miR-320a, miR-25, miR-126, miR-19b, miR-199a-3p, miR-30b, miR-30c, miR-374a) that can be used to create a reliable reference combination of 3 miRNAs. We showed that on average the mean of 3 miRNAs (p = 0.0002) and 2 miRNAs (p = 0.0031) were a better reference than single miRNA. The arithmetic means of 3 miRNAs: miR-24, miR-222 and miR-27a was shown to be the most stable combination of 3 miRNAs in validation sets. CONCLUSIONS: No single miRNA was suitable as a universal reference in serum miRNA qPCR profiling, but it was possible to designate a set of miRNAs, which consistently contributed to most stable combinations.
Asunto(s)
MicroARN Circulante/genética , Biología Computacional/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Algoritmos , Simulación por Computador , Bases de Datos Genéticas , Perfilación de la Expresión Génica/normas , Humanos , Estándares de ReferenciaRESUMEN
Expression of BRCA1 is commonly decreased in sporadic breast tumors, and this correlates with poor prognosis of breast cancer patients. Here we show that BRCA1 transcripts are selectively enriched in the Argonaute/miR-182 complex and miR-182 downregulates BRCA1 expression. Antagonizing miR-182 enhances BRCA1 protein levels and protects them from IR-induced cell death, while overexpressing miR-182 reduces BRCA1 protein, impairs homologous recombination-mediated repair, and render cells hypersensitive to IR. The impaired DNA repair phenotype induced by miR-182 overexpression can be fully rescued by overexpressing miR-182-insensitive BRCA1. Consistent with a BRCA1-deficiency phenotype, miR-182-overexpressing breast tumor cells are hypersensitive to inhibitors of poly (ADP-ribose) polymerase 1 (PARP1). Conversely, antagonizing miR-182 enhances BRCA1 levels and induces resistance to PARP1 inhibitor. Finally, a clinical-grade PARP1 inhibitor impacts outgrowth of miR-182-expressing tumors in animal models. Together these results suggest that miR-182-mediated downregulation of BRCA1 impedes DNA repair and may impact breast cancer therapy.
Asunto(s)
Antineoplásicos/farmacología , Proteína BRCA1/genética , Reparación del ADN/efectos de los fármacos , MicroARNs/fisiología , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Animales , Diferenciación Celular , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de la radiación , Regulación hacia Abajo , Humanos , Células K562 , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Poli(ADP-Ribosa) Polimerasa-1RESUMEN
BACKGROUND: Senescence is a fundamental biological process implicated in various pathologies, including cancer. Regarding carcinogenesis, senescence signifies, at least in its initial phases, an anti-tumor response that needs to be circumvented for cancer to progress. Micro-RNAs, a subclass of regulatory, non-coding RNAs, participate in senescence regulation. At the subcellular level micro-RNAs, similar to proteins, have been shown to traffic between organelles influencing cellular behavior. The differential function of micro-RNAs relative to their subcellular localization and their role in senescence biology raises concurrent in situ analysis of coding and non-coding gene products in senescent cells as a necessity. However, technical challenges have rendered in situ co-detection unfeasible until now. METHODS: In the present report we describe a methodology that bypasses these technical limitations achieving for the first time simultaneous detection of both a micro-RNA and a protein in the biological context of cellular senescence, utilizing the new commercially available SenTraGorTM compound. The method was applied in a prototypical human non-malignant epithelial model of oncogene-induced senescence that we generated for the purposes of the study. For the characterization of this novel system, we applied a wide range of cellular and molecular techniques, as well as high-throughput analysis of the transcriptome and micro-RNAs. RESULTS: This experimental setting has three advantages that are presented and discussed: i) it covers a "gap" in the molecular carcinogenesis field, as almost all corresponding in vitro models are fibroblast-based, even though the majority of neoplasms have epithelial origin, ii) it recapitulates the precancerous and cancerous phases of epithelial tumorigenesis within a short time frame under the light of natural selection and iii) it uses as an oncogenic signal, the replication licensing factor CDC6, implicated in both DNA replication and transcription when over-expressed, a characteristic that can be exploited to monitor RNA dynamics. CONCLUSIONS: Consequently, we demonstrate that our model is optimal for studying the molecular basis of epithelial carcinogenesis shedding light on the tumor-initiating events. The latter may reveal novel molecular targets with clinical benefit. Besides, since this method can be incorporated in a wide range of low, medium or high-throughput image-based approaches, we expect it to be broadly applicable.
Asunto(s)
Senescencia Celular/genética , Neoplasias Glandulares y Epiteliales/genética , Oncogenes , Carcinogénesis , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Genoma , Humanos , MicroARNs/metabolismo , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Glandulares y Epiteliales/ultraestructura , Proteínas Nucleares/metabolismo , Proteínas/metabolismoRESUMEN
The involvement of whole-chromosome aneuploidy in tumorigenesis is the subject of debate, in large part because of the lack of insight into underlying mechanisms. Here we identify a mechanism by which errors in mitotic chromosome segregation generate DNA breaks via the formation of structures called micronuclei. Whole-chromosome-containing micronuclei form when mitotic errors produce lagging chromosomes. We tracked the fate of newly generated micronuclei and found that they undergo defective and asynchronous DNA replication, resulting in DNA damage and often extensive fragmentation of the chromosome in the micronucleus. Micronuclei can persist in cells over several generations but the chromosome in the micronucleus can also be distributed to daughter nuclei. Thus, chromosome segregation errors potentially lead to mutations and chromosome rearrangements that can integrate into the genome. Pulverization of chromosomes in micronuclei may also be one explanation for 'chromothripsis' in cancer and developmental disorders, where isolated chromosomes or chromosome arms undergo massive local DNA breakage and rearrangement.
Asunto(s)
Aneuploidia , Rotura Cromosómica , Micronúcleos con Defecto Cromosómico , Mitosis , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Segregación Cromosómica , Ensayo Cometa , Fragmentación del ADN , Replicación del ADN , Humanos , Mitosis/genética , Neoplasias/etiología , Neoplasias/genética , Neoplasias/patologíaRESUMEN
miR-24, upregulated during terminal differentiation of multiple lineages, inhibits cell-cycle progression. Antagonizing miR-24 restores postmitotic cell proliferation and enhances fibroblast proliferation, whereas overexpressing miR-24 increases the G1 compartment. The 248 mRNAs downregulated upon miR-24 overexpression are highly enriched for DNA repair and cell-cycle regulatory genes that form a direct interaction network with prominent nodes at genes that enhance (MYC, E2F2, CCNB1, and CDC2) or inhibit (p27Kip1 and VHL) cell-cycle progression. miR-24 directly regulates MYC and E2F2 and some genes that they transactivate. Enhanced proliferation from antagonizing miR-24 is abrogated by knocking down E2F2, but not MYC, and cell proliferation, inhibited by miR-24 overexpression, is rescued by miR-24-insensitive E2F2. Therefore, E2F2 is a critical miR-24 target. The E2F2 3'UTR lacks a predicted miR-24 recognition element. In fact, miR-24 regulates expression of E2F2, MYC, AURKB, CCNA2, CDC2, CDK4, and FEN1 by recognizing seedless but highly complementary sequences.