RESUMEN

Organisms have evolved a complex system, called the DNA damage response (DDR), which maintains genome integrity. The DDR is responsible for identifying and repairing a variety of lesions and alterations in DNA. DDR proteins coordinate DNA damage detection, cell cycle arrest, and repair, with many of these events regulated by protein phosphorylation. In the human proteome, 23 proteins contain the BRCT (BRCA1 C-Terminus domain) domain, a modular signaling domain that can bind phosphopeptides and mediate protein-protein interactions. BRCTs can be found as functional single units, tandem (tBRCT), triplet (tpBRCT), and quartet. Here we examine the evolution of the tpBRCT architecture present in TOPBP1 (DNA topoisomerase II binding protein 1) and ECT2 (epithelial cell transforming 2), and their respective interaction partners RAD9 (Cell cycle checkpoint control protein RAD9) and CYK-4 (Rac GTPase-activating protein 1), with a focus on the conservation of the phosphopeptide-binding residues. The pair TOPBP1-RAD9 arose with the Eukaryotes and ECT2-CYK-4 with the Eumetazoans. Triplet structural and functional characteristics were conserved in almost all organisms. The first unit of the triplet (BRCT0) is different from the other two BRCTs but conserved between orthologs for both TOPBP1 and ECT2. BRCT domain evolution simulations suggest a trend to retain the singlet or towards two or three BRCT copies per protein consistent with functional tBRCT and tpBRCT architectures. Our results shed light on the emergence of the function and architecture of multiple BRCT domain organizations and provide information about the evolution of the BRCT triplet. Knowledge of BRCT domain evolution can improve the understanding of DNA damage response mechanisms and signal transduction in DDR.

Asunto(s)

Proteína BRCA1 , Proteínas de Ciclo Celular , Humanos , Proteína BRCA1/metabolismo , Dominios Proteicos , Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Transducción de Señal , Fosforilación , Unión Proteica

RESUMEN

BRCA1 (Breast Cancer 1, early onset) is linked to breast and ovarian cancer predisposition. Still, the risks conferred by a significant portion of BRCA1 variants identified in the population remains unknown. Most of these variants of uncertain significance are missense alterations. However, the functional implications of small in-frame deletions and/or insertions (indels) are also difficult to predict. Our group has previously evaluated the functional impact of 347 missense variants using an extensively validated transcriptional activity assay. Here we show a systematic assessment of 30 naturally occurring in-frame indels located at the C-terminal region of BRCA1. We identified positions sensitive and tolerant to alterations, expanding the knowledge of structural determinants of BRCA1 function. We further designed and assessed the impact of four single codon deletions in the tBRCT linker region and six nonsense variants at the C-terminus end of BRCA1. Amino acid substitutions, deletions or insertions in the disordered region do not significantly impact activity and are not likely to constitute pathogenic alleles. On the other hand, a sizeable fraction of in-frame indels at the BRCT domain significantly impact function. We then use a Bayesian integrative statistical model to derive the probability of pathogenicity for each variant. Our data highlights the importance of assessing the impact of small in-frame indels in BRCA1 to improve risk assessment and clinical decisions for carriers.

Asunto(s)

Neoplasias de la Mama , Neoplasias Ováricas , Alelos , Sustitución de Aminoácidos , Proteína BRCA1/metabolismo , Teorema de Bayes , Femenino , Genes BRCA1 , Predisposición Genética a la Enfermedad , Humanos , Mutación Missense , Neoplasias Ováricas/genética

RESUMEN

The BRCA1-A complex contains matching lysine-63 ubiquitin (K63-Ub) binding and deubiquitylating activities. How these functionalities are coordinated to effectively respond to DNA damage remains unknown. We generated Brcc36 deubiquitylating enzyme (DUB) inactive mice to address this gap in knowledge in a physiologic system. DUB inactivation impaired BRCA1-A complex damage localization and repair activities while causing early lethality when combined with Brca2 mutation. Damage response dysfunction in DUB-inactive cells corresponded to increased K63-Ub on RAP80 and BRCC36. Chemical cross-linking coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and cryogenic-electron microscopy (cryo-EM) analyses of isolated BRCA1-A complexes demonstrated the RAP80 ubiquitin interaction motifs are occupied by ubiquitin exclusively in the DUB-inactive complex, linking auto-inhibition by internal K63-Ub chains to loss of damage site ubiquitin recognition. These findings identify RAP80 and BRCC36 as autologous DUB substrates in the BRCA1-A complex, thus explaining the evolution of matching ubiquitin-binding and hydrolysis activities within a single macromolecular assembly.

Asunto(s)

Proteína BRCA1 , Daño del ADN , Proteínas de Unión al ADN , Enzimas Desubicuitinizantes , Chaperonas de Histonas , Animales , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Cromatografía Liquida , Reparación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/metabolismo , Células HeLa , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Humanos , Ratones , Espectrometría de Masas en Tándem , Ubiquitina/metabolismo

RESUMEN

Single nucleotide variants (SNVs) occurring in a protein coding gene may disrupt its function in multiple ways. Predicting this disruption has been recognized as an important problem in bioinformatics research. Many tools, hereafter p-tools, have been designed to perform these predictions and many of them are now of common use in scientific research, even in clinical applications. This highlights the importance of understanding the semantics of their outputs. To shed light on this issue, two questions are formulated, (i) do p-tools provide similar predictions? (inner consistency), and (ii) are these predictions consistent with the literature? (outer consistency). To answer these, six p-tools are evaluated with exhaustive SNV datasets from the BRCA1 gene. Two indices, called K a l l and K s t r o n g , are proposed to quantify the inner consistency of pairs of p-tools while the outer consistency is quantified by standard information retrieval metrics. While the inner consistency analysis reveals that most of the p-tools are not consistent with each other, the outer consistency analysis reveals they are characterized by a low prediction performance. Although this result highlights the need of improving the prediction performance of individual p-tools, the inner consistency results pave the way to the systematic design of truly diverse ensembles of p-tools that can overcome the limitations of individual members.

Asunto(s)

Proteína BRCA1 , Biología Computacional , Modelos Genéticos , Polimorfismo de Nucleótido Simple , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Humanos

RESUMEN

BACKGROUND: Homologous recombination deficiencies are associated with increased platinum sensitivity and potential response to poly (ADP-ribose) polymerase inhibitors in epithelial ovarian cancer. As an alternative to germline testing or somatic tumor sequencing, BRCA1 deficiency can be detected by immunohistochemistry and might predict homologous recombination deficiencies. OBJECTIVE: This study aimed to assess the association between BRCA1 expression by immunohistochemistry and the prognosis of patients with epithelial ovarian cancer. METHODS: We conducted a systematic review and meta-analysis following the Preferred Reporting Items for Systematic reviews and Meta-Analyses statement. We searched PubMed, EMBASE, Web of Science, and Scopus databases through July 2019. Reference lists of selected articles were screened for further studies. We conducted qualitative synthesis and meta-analyses of hazard ratios for overall survival and progression-free survival. RESULTS: Of 41 studies of BRCA1 expression using immunohistochemistry, 18 evaluated the association of BRCA1 expression with patient survival (2738 cases). The loss of BRCA1 expression was associated with improved overall survival (hazard ratio = 0.67, 95% confidence interval 0.57-0.77) and progression-free survival (hazard ratio = 0.70, 95% confidence interval 0.58-0.84). CONCLUSIONS: Negative BRCA1 expression assessed by immunohistochemistry was associated with a better prognosis in epithelial ovarian cancer.

Asunto(s)

Proteína BRCA1/metabolismo , Inmunohistoquímica/métodos , Neoplasias Ováricas/tratamiento farmacológico , Femenino , Humanos , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/mortalidad , Pronóstico , Supervivencia sin Progresión

RESUMEN

BACKGROUND: Loss of function in either breast cancer type 1 susceptibility protein (BRCA1) or breast cancer type 2 susceptibility protein (BRCA2) is a major risk factor for epithelial ovarian cancer (EOC) development. BRCA1 or BRCA2 deficiencies are associated with short-term prognosis and might have importance for the treatment of women with the disease. However, the screening of all possible mechanisms of dysfunction is expensive, time-consuming and difficult to apply in clinical practice. On the other hand, immunohistochemistry (IHC) is a simple and reliable method to access the expression of several proteins in tumour tissues. MATERIALS AND METHODS: This systematic review aims to evaluate the current usage of IHC to detect BRCA1 and BRCA2 deficiencies in EOC. We searched and evaluated all primary literature on the use of IHC for evaluating BRCA1 and BRCA2 proteins expression in EOC. The main concepts for the search were: ovarian neoplasms, IHC, BRCA1 and BRCA2. RESULTS: Forty-four studies from 925 unique titles were included. A total of 4206 tumour samples were evaluated for BRCA1 and 1041 for BRCA2 expression. Twelve BRCA1 primary antibodies were used in 41 studies, and the most common was the MS110 clone (75.6%). Seven BRCA2 primary antibodies were used in ten studies. Using the cut-off of 10%, 47.0% of EOCs are associated with loss of BRCA1 and 34.5% with the loss of BRCA2 expression. CONCLUSION: IHC was effective to detect loss of BRCA1 protein expression in EOC; however, data on BRCA2 expression were heterogeneous and difficult to interpret.

Asunto(s)

Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Neoplasias Ováricas/mortalidad , Anticuerpos/metabolismo , Proteína BRCA1/inmunología , Proteína BRCA2/inmunología , Femenino , Humanos , Inmunohistoquímica , Factores de Riesgo

RESUMEN

BRCA-1 is a nuclear protein involved in DNA repair, transcriptional regulation, and cell cycle control. Its involvement in other cellular processes has been described. Here, we aimed to investigate the role of BRCA-1 in macrophages M(LPS), M(IL-4), and tumor cell-induced differentiation. We used siRNAs to knockdown BRCA-1 in RAW 264.7 macrophages exposed to LPS, IL-4, and C6 glioma cells conditioned medium (CMC6), and evaluated macrophage differentiation markers and functional phagocytic activity as well as DNA damage and cell survival in the presence and absence of BRCA-1. LPS and CMC6, but not by IL-4, increased DNA damage in macrophages, and this effect was more pronounced in BRCA-1-depleted cells, including M(IL-4). BRCA-1 depletion impaired expression of pro-inflammatory cytokines, TNF-α and IL-6, and reduced the phagocytic activity of macrophages in response to LPS. In CMC6-induced differentiation, BRCA-1 knockdown inhibited TNF-α and IL-6 expression which was accompanied by upregulation of the anti-inflammatory markers IL-10 and TGF-ß and reduced phagocytosis. In contrast, M(IL-4) phenotype was not affected by BRCA-1 status. Molecular docking predicted that the conserved BRCA-1 domain BRCT can interact with the p65 subunit of NF-κB. Immunofluorescence assays showed that BRCA-1 and p65 co-localize in the nucleus of LPS-treated macrophages and reporter gene assay showed that depletion of BRCA-1 decreased LPS and CMC6-induced NF-κB transactivation. IL-4 had no effect upon NF-κB. Taken together, our findings suggest a role of BRCA-1 in macrophage differentiation and phagocytosis induced by LPS and tumor cells secretoma, but not IL-4, in a mechanism associated with inhibition of NF-κB.

Asunto(s)

Proteína BRCA1/metabolismo , Polaridad Celular , Inflamación/patología , Activación de Macrófagos , Macrófagos/metabolismo , Macrófagos/patología , FN-kappa B/metabolismo , Animales , Biomarcadores/metabolismo , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Polaridad Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Daño del ADN , Inflamación/metabolismo , Lipopolisacáridos , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Metaloproteinasas de la Matriz/metabolismo , Ratones , Fagocitosis/efectos de los fármacos , Células RAW 264.7 , ARN Interferente Pequeño/metabolismo , Ratas

RESUMEN

Epithelial ovarian cancer is a serious public health problem worldwide with the highest mortality rate of all gynecologic cancers. The current standard-of-care for the treatment of ovarian cancer is based on chemotherapy based on adjuvant cisplatin/carboplatin and taxane regimens that represent the first-line agents for patients with advanced disease. The DNA repair activity of cancer cells determines the efficacy of anticancer drugs. These features make DNA repair mechanisms a promising target for novel cancer treatments. In this context a better understanding of the DNA damage response caused by antitumor agents has provided the basis for the use of DNA repair inhibitors to improve the therapeutic use of DNA-damaging drugs. In this review, we will discuss the functions of DNA repair proteins and the advances in targeting DNA repair pathways with special emphasis in the inhibition of HRR and BER in ovarian cancer. We focused in the actual efforts in the development and clinical use of poly (ADPribose) polymerase (PARP) inhibitors for the intervention of BRCA1/BRCA2-deficient ovarian tumors. The clinical development of PARP inhibitors in ovarian cancer patients with germline BRCA1/2 mutations and sporadic high-grade serous ovarian cancer is ongoing. Some phase II and phase III trials have been completed with promising results for ovarian cancer patients.

Asunto(s)

Antineoplásicos/farmacología , Reparación del ADN/efectos de los fármacos , Terapia Molecular Dirigida , Neoplasias Ováricas , Antineoplásicos/uso terapéutico , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Femenino , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico

RESUMEN

Mutations in FMS-like tyrosine kinase 3 (FLT3), such as internal tandem duplications (ITDs), can be found in up to 23% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. Current treatment options for FLT3(ITD)-positive AMLs include genotoxic therapy and FLT3 inhibitors (FLT3i's), which are rarely curative. PARP1 inhibitors (PARP1i's) have been successfully applied to induce synthetic lethality in tumors harboring BRCA1/2 mutations and displaying homologous recombination (HR) deficiency. We show here that inhibition of FLT3(ITD) activity by the FLT3i AC220 caused downregulation of DNA repair proteins BRCA1, BRCA2, PALB2, RAD51, and LIG4, resulting in inhibition of 2 major DNA double-strand break (DSB) repair pathways, HR, and nonhomologous end-joining. PARP1i, olaparib, and BMN673 caused accumulation of lethal DSBs and cell death in AC220-treated FLT3(ITD)-positive leukemia cells, thus mimicking synthetic lethality. Moreover, the combination of FLT3i and PARP1i eliminated FLT3(ITD)-positive quiescent and proliferating leukemia stem cells, as well as leukemic progenitors, from human and mouse leukemia samples. Notably, the combination of AC220 and BMN673 significantly delayed disease onset and effectively reduced leukemia-initiating cells in an FLT3(ITD)-positive primary AML xenograft mouse model. In conclusion, we postulate that FLT3i-induced deficiencies in DSB repair pathways sensitize FLT3(ITD)-positive AML cells to synthetic lethality triggered by PARP1i's. Therefore, FLT3(ITD) could be used as a precision medicine marker for identifying AML patients that may benefit from a therapeutic regimen combining FLT3 and PARP1i's.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Reparación del ADN/efectos de los fármacos , Leucemia Mieloide Aguda , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/metabolismo , Animales , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Benzotiazoles/farmacología , Línea Celular Tumoral , ADN Ligasa (ATP)/genética , ADN Ligasa (ATP)/metabolismo , Proteína del Grupo de Complementación N de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación N de la Anemia de Fanconi/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Mutación , Compuestos de Fenilurea/farmacología , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa 3 Similar a fms/genética

RESUMEN

There are many factors which make canine cancer like cancer in humans. The occurrence of spontaneous mammary tumors in pet dogs, tumor genetics, molecular targets and exposure to the same environmental risk factors are among these factors. Therefore, the study of canine cancer can provide useful information to the oncology field. This study aimed to establish and characterize a panel of primary mixed cell cultures obtained from spontaneous canine mammary tumors. Eight established cell cultures obtained from one normal mammary gland, one complex adenoma, one mixed adenoma, two complex carcinomas and two mixed carcinomas were analyzed. The gene expression levels of classic molecular cancer players such as fibroblast growth factor receptor (FGFR) 2, breast cancer (BRCA) 1, BRCA2 and estrogen receptor (ESR) 1 were evaluated. For the first time, three orphan nuclear receptors, estrogen-related receptors (ERRs) α, ß and γ were studied in canine mammary cancer. The highest expression level of ERRα was observed in complex carcinoma-derived cell culture, while the highest levels of ERRß and γ were observed in cells derived from a mixed carcinoma. Meanwhile, complex carcinomas presented the highest levels of expression of ESR1, BRCA1 and FGFR2 among all samples. BRCA2 was found exclusively in complex adenoma. The transcription factor GATA3 had its highest levels in mixed carcinoma samples and its lowest levels in complex adenoma. Proliferation assays were also performed to evaluate the mixed cell cultures response to ER ligands, genistein and DES, both in normoxia and hypoxic conditions. Our results demonstrate that morphological and functional studies of primary mixed cell cultures derived from spontaneous canine mammary tumors are possible and provide valuable tool for the study of various stages of mammary cancer development.

Asunto(s)

Enfermedades de los Perros/metabolismo , Neoplasias Mamarias Animales/metabolismo , Animales , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Perros , Receptor alfa de Estrógeno/metabolismo , Femenino , Citometría de Flujo , Ploidias , Cultivo Primario de Células , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo

RESUMEN

The heteromorphic X and Y chromosomes behave in a special way in mammalian spermatocytes; they form the XY body and synapse only partially. The aim of this article was to study the origin and the role of the special differentiations in the XY pair of the domestic cat during pachytene by analyzing its fine structural characteristics and the immunolocalization of the main meiotic proteins SYCP3, SYCP1, SYCE3, SMC3, γ-H2AX, BRCA1, H3K27me3, and MLH1. The cat XY body shows particularly striking structures: an extreme degree of axial fibrillation in late pachynema and a special location of SYCP3-containing fibrils, bridging different regions of the main X axis, as well as one bridge at the inner end of the pairing region that colocalizes with the single mandatory MLH1 focus. There are sequential changes, first bullous expansions, then subdivision into fibrils, all involving axial thickening. The chromatin of the XY body presents the usual features of meiotic sex chromosome inactivation. An analysis of the XY body of many eutherians and metatherians suggests that axial thickenings are primitive features. The sequential changes in the mass and location of SYCP3-containing fibers vary among the clades because of specific processes of axial assembly/disassembly occurring in different species.

Asunto(s)

Gatos/genética , Proteínas Nucleares/metabolismo , Fase Paquiteno/genética , Complejo Sinaptonémico/metabolismo , Cromosoma X/metabolismo , Cromosoma X/ultraestructura , Cromosoma Y/metabolismo , Cromosoma Y/ultraestructura , Animales , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Cromatina/metabolismo , Cromatina/ultraestructura , Histonas/genética , Histonas/metabolismo , Masculino , Microscopía Fluorescente , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , Espermatocitos/metabolismo , Complejo Sinaptonémico/genética

RESUMEN

Double strand break lesions, the most toxic type of DNA damage, are repaired primarily through 2 distinct pathways: homology-directed recombination (HR) and non-homologous end-joining (NHEJ). BRCA1 and 53BP1, 2 proteins containing the BRCT modular domain, play an important role in DNA damage response (DDR) by orchestrating the decision between HR and NHEJ, but the precise mechanisms regarding both pathways are not entirely understood. Previously, our group identified a putative interaction between BRCA1 and BARD1 (BRCA1-associated RING domain 1) and the cyclin-dependent kinase (CDK9). CDK9 is a component of the positive transcription elongation complex and has been implicated in genome integrity maintenance associated with the replication stress response. Here we show that CDK9 interacts with endogenous BRCA1 and BARD1 mediated by their RING finger and BRCT domains, and describe CDK9 ionizing radiation-induced foci (IRIF) formation and its co-localization with BRCA1 in DNA damage sites. Cells lacking CDK9 are characterized by an altered γ-H2AX foci dynamics after DNA damage, a reduced efficiency in HR but not in NHEJ repair, failure to form BRCA1 and RAD51 IRIF and increased sensitivity to genotoxic agents. These data indicate that CDK9 is a player in the DDR and is consistent with its participation in HR pathway by modulating BRCA1 response.

Asunto(s)

Proteína BRCA1/metabolismo , Quinasa 9 Dependiente de la Ciclina/metabolismo , Daño del ADN , Roturas del ADN de Doble Cadena/efectos de la radiación , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Mutágenos/toxicidad , Unión Proteica/efectos de la radiación , ARN Interferente Pequeño/metabolismo , Recombinasa Rad51/metabolismo , Radiación Ionizante , Proteínas Supresoras de Tumor/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo

RESUMEN

The Aurora protein kinase (AURKA) and the Polo-like kinase-1 (PLK1) activate the cell cycle, and they are considered promising druggable targets in cancer therapy. However, resistance to chemotherapy and to specific small­molecule inhibitors is common in cancer patients; thus alternative therapeutic approaches are needed to overcome clinical resistance. Here, we showed that the dietary compound resveratrol suppressed the cell cycle by targeting AURKA and PLK1 kinases. First, we identified genes modulated by resveratrol using a genome-wide analysis of gene expression in MDA-MB-231 breast cancer cells. Transcriptional profiling indicated that 375 genes were modulated at 24 h after resveratrol intervention, whereas 579 genes were regulated at 48 h. Of these, 290 genes were deregulated in common at 24 and 48 h. Interestingly, a significant decrease in the expression of genes involved in the cell cycle, DNA repair, cytoskeleton organization, and angiogenesis was detected. In particular, AURKA and PLK1 kinases were downregulated by resveratrol at 24 h. In addition the BRCA1 gene, an AURKA/PLK1 inhibitor, was upregulated at 24 h of treatment. Moreover, two well-known resveratrol effectors, cyclin D1 (CCND1) and cyclin B1 (CCNB1), were also repressed at both times. Congruently, we found that resveratrol impaired G1/S phase transition in both MDA-MB-231 and MCF-7 cells. By western blot assays, we confirmed that resveratrol suppressed AURKA, CCND1 and CCNB1 at 24 and 48 h. In summary, we showed for the first time that resveratrol regulates cell cycle progression by targeting AURKA and PLK1. Our findings highlight the potential use of resveratrol as an adjuvant therapy for breast cancer.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Aurora Quinasa A/antagonistas & inhibidores , Proteína BRCA1/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Estilbenos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Ciclina B1/antagonistas & inhibidores , Ciclina D1/antagonistas & inhibidores , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Células MCF-7 , Análisis de Secuencia por Matrices de Oligonucleótidos , Resveratrol , Transcriptoma/genética , Quinasa Tipo Polo 1

RESUMEN

BRCA1 has been found to be absent or miss localized in the cytoplasm in a relevant proportion of breast cancer tumors with no germline mutations. BRCA1 main function is in the nucleus, and its interaction with BARD1 is relevant for its nuclear translocation and retention. Our aim was to analyze the sub-cellular localization of BRCA1 and BARD1 in breast cancer tumors, and determine the level of expression of their splice variants BRCA1-Δ11q and BARD1-α and BARD1-ß. BRCA1 and BARD1 expressions were performed by immunohistochemistry and immunofluorescence in 103 breast cancer tumors. Colocalization was determined by confocal microscopy. Transcript variants were determined by qRT-PCR. We found BRCA1 localized in the cytoplasm with BARD1 in 51.4 % of tumors. An exclusive nuclear localization of both proteins was observed in 7/103 tumors (6.8 %). Indeed, these tumors displayed an apparent nucleolar colocalization of BARD1 and BRCA1. In relation to splice variants, there is a tendency to an overexpression of BARD1-α mRNA (30 % of tumors) and a decreased expression of BARD1-ß (41 %). BRCA1 full-length was downregulated in 63 % of tumors, and 37 % showed BRCA1-Δ11q variant overexpressed. Our findings contribute to a better understanding of the expression and sub-cellular localization of BRCA1 in breast cancer tumors. Interaction of BRCA1 and BARD1 seems to be not affected in 58.2 % of tumors, which showed colocalization of both proteins. The absence of BRCA1 in 41 % of tumors reveals a BRCAness phenotype, constituting an excellent marker for therapy sensitivity, to platinum drugs or PARP inhibitors.

Asunto(s)

Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Empalme Alternativo , Neoplasias de la Mama/patología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Femenino , Humanos , Unión Proteica , Isoformas de Proteínas , Transporte de Proteínas

RESUMEN

Germline mutations in identified breast cancer susceptibility genes account for less than 20% of Chinese familial breast cancers. Dicer is an essential component of the microRNA-producing machinery; germline mutations of DICER1 have been confirmed in familial pleuropulmonary blastoma, ovarian sex cord-stromal tumors, and other cancers. Low expression of DICER1 is frequently detected in breast cancer. However, whether germline mutations of DICER1 occur in familial breast cancers remain unknown. Sixty-five breast cancer probands from BRCA1/BRCA2-negative Chinese breast cancer families were screened for germline mutations in DICER1. In addition, 100 unrelated healthy females were enrolled as controls. A polymerase chain reaction sequencing assay was used to screen for mutations in coding regions and at the exon-intron boundaries of DICER1. All variants in introns were evaluated using the NNSplice software to determine the potential splicing effect. A total of 12 germline variants were found, including 11 variants in introns and 1 variant in the 3'-non-coding region. Four variants (IVS8-205 C>T, IVS11+131 delGAAA, IVS16+42 delTA, and IVS19+160 T>C) were novel. Three variants (IVS11+105 C>T, IVS16+42 delTA, and 6095 T>A) may affect splice sites. None of the observed variants appeared to be disease-related, suggesting that germline mutations in DICER1 are rare or absent in familial breast cancer patients.

Asunto(s)

ARN Helicasas DEAD-box/genética , Mutación de Línea Germinal , Neoplasias Ováricas/genética , Blastoma Pulmonar/genética , Ribonucleasa III/genética , Tumores de los Cordones Sexuales y Estroma de las Gónadas/genética , Adulto , Anciano , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , China , Simulación por Computador , ARN Helicasas DEAD-box/metabolismo , Femenino , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/metabolismo , Isoformas de Proteínas/metabolismo , Blastoma Pulmonar/metabolismo , Ribonucleasa III/metabolismo , Tumores de los Cordones Sexuales y Estroma de las Gónadas/metabolismo , Adulto Joven

RESUMEN

This study aimed to evaluate the association between RRM1 and BRCA1 expressions and the therapeutic efficacy of platinum-based chemotherapy in non-small cell lung cancer patients in terms of their response and prognosis. In total, 377 patients agreed to participate in our study, and all of them received platinum-based combination chemotherapy between January 2008 and January 2009. The relative cDNA quantitation for RRM1 and BRCA1 was conducted using a fluorescence-based, real-time detection method, using ß-actin as a reference gene. The average age of the 377 patients was 64.6 years (range: 25.5-86.4 years), including 269 men and 108 women. Patients with high RRM1 expression benefited more from a platinum-containing regimen, and patients with high BRCA1 expression showed a high response rate to a platinum-containing regimen and reduced disease progression. Patients with high RRM1 expression were associated with a longer progression-free survival (PFS) and overall survival (OS) than those with low expression, and the hazard ratios (HRs) (95% confidence interval (CI)) were 0.67 (0.32-0.91) and 0.54 (0.30-0.95), respectively. Patients with high BRCA1 expression showed longer PFS and OS compared to those with low expression, and the HRs (95%CI) were 0.54 (0.30-0.95) and 0.62 (0.32-0.93), respectively. These results could be used in personalized chemotherapy decisions and to increase the response rate and prolonged survival, and could encourage exploration of the predictive value of other genes.

Asunto(s)

Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica , Proteína BRCA1/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , ARN Mensajero/genética , Proteínas Supresoras de Tumor/genética , Adenocarcinoma/genética , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Proteína BRCA1/metabolismo , Biomarcadores Farmacológicos/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Cisplatino/administración & dosificación , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Femenino , Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Paclitaxel/administración & dosificación , ARN Mensajero/metabolismo , Ribonucleósido Difosfato Reductasa , Análisis de Supervivencia , Resultado del Tratamiento , Proteínas Supresoras de Tumor/metabolismo , Vinblastina/administración & dosificación , Vinblastina/análogos & derivados , Vinorelbina , Gemcitabina

RESUMEN

Germline inactivating variants in BRCA1 lead to a significantly increased risk of breast and ovarian cancers in carriers. While the functional effect of many variants can be inferred from the DNA sequence, determining the effect of missense variants present a significant challenge. A series of biochemical and cell biological assays have been successfully used to explore the impact of these variants on the function of BRCA1, which contribute to assessing their likelihood of pathogenicity. It has been determined that variants that co-localize with structural or functional motifs are more likely to disrupt the stability and function of BRCA1. Here we assess the functional impact of 37 variants chosen to probe the functional impact of variants in phosphorylation sites and in the BRCT domains. In addition, we perform a meta-analysis of 170 unique variants tested by the transcription activation assays in the carboxy-terminal domain of BRCA1 using a recently developed computation model to provide assessment for functional impact and their likelihood of pathogenicity.

Asunto(s)

Proteína BRCA1/química , Proteína BRCA1/metabolismo , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad , Alelos , Sustitución de Aminoácidos , Proteína BRCA1/genética , Teorema de Bayes , Secuencia Conservada , Humanos , Mutación Missense , Dominios y Motivos de Interacción de Proteínas/genética , Termodinámica

RESUMEN

Laser phototherapy (LPT) is widely used in clinical practice to accelerate healing. Although the use of LPT has advantages, the molecular mechanisms involved in the process of accelerated healing and the safety concerns associated with LPT are still poorly understood. We investigated the physiological effects of LPT irradiation on the production and accumulation of reactive oxygen species (ROS), genomic instability, and deoxyribose nucleic acid (DNA) damage in human epithelial cells. In contrast to a high energy density (20 J/cm²), laser administered at a low energy density (4 J/cm²) resulted in the accumulation of ROS. Interestingly, 4 J/cm² of LPT did not induce DNA damage, genomic instability, or nuclear influx of the BRCA1 DNA damage repair protein, a known genome protective molecule that actively participates in DNA repair. Our results suggest that administration of low energy densities of LPT induces the accumulation of safe levels of ROS, which may explain the accelerated healing results observed in patients. These findings indicate that epithelial cells have an endowed molecular circuitry that responds to LPT by physiologically inducing accumulation of ROS, which triggers accelerated healing. Importantly, our results suggest that low energy densities of LPT can serve as a safe therapy to accelerate epithelial healing.

Asunto(s)

Roturas del ADN de Doble Cadena/efectos de la radiación , Células Epiteliales/efectos de la radiación , Terapia por Luz de Baja Intensidad , Especies Reactivas de Oxígeno/metabolismo , Proteína BRCA1/análisis , Proteína BRCA1/metabolismo , Línea Celular , Reparación del ADN , Células Epiteliales/metabolismo , Histonas/análisis , Histonas/metabolismo , Humanos , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/efectos de la radiación

RESUMEN

Bladder cancer is a common cancer worldwide. For patients presenting with muscle-invasive disease, the 5-year survival rate is approximately 50%. Cisplatin-based combination chemotherapy is recommended in the neoadjuvant setting before cystectomy and is also the first line in the metastatic setting. However, the survival benefit of such therapy is modest. The identification of pharmacogenomic biomarkers would enable the rational and personalized treatment of patients by selecting those patients that would benefit most from such therapies sparing others the unnecessary toxicity. Conventional therapies would be recommended for an expected responder, whereas a nonresponder would be considered for alternative therapies selected on the basis of the individual's molecular profile. Although few effective bladder cancer therapies have been introduced in the past 30 years, several targeted therapies against the molecular drivers of bladder cancer appear promising. This review summarizes pharmacogenomic biomarkers that require further investigation or prospective evaluation or both, and publicly available tools for drug discovery and biomarker identification from in vitro data.

Asunto(s)

Farmacogenética/métodos , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Antineoplásicos/farmacología , Proteína BRCA1/metabolismo , Biomarcadores de Tumor/metabolismo , Cisplatino/farmacología , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Humanos , Terapia Neoadyuvante , Estudios Prospectivos , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

BACKGROUND: Loss or mutations of the BRCA1 gene are associated with increased risk of breast and ovarian cancers and with prostate cancer (PCa) aggressiveness. Previously, we identified GADD153 as a target of BRCA1 protein, which increases doxorubicin sensitivity in human p53 -/- PCa cells (PC3). Considering that p53 is a crucial target in cancer therapy, in this work we investigated p53 role in the regulation of transcription of GADD153. METHODS: We performed reverse transcription quantitative PCR (RT-qPCR), western blot and luciferase assays to analyze GADD153 and/or BRCA1 expression in response to ultraviolet or doxorubicin exposure in PC3 p53 stable-transfected cells and LNCaP (p53+/+) cells. BRCA1 protein recruitment to GADD153 promoter was studied by chromatin immunoprecipitation-qPCR. To assess expression of BRCA1 and/or p53 target genes, we used a panel of stable-transfected PCa cell lines. We finally analyzed these genes in vivo using BRCA1-depleted PCa xenograft models. RESULTS: We found that GADD153 was highly induced by doxorubicin in PC3 cells; however, this response was totally abolished in LNCaP (p53wt) and in p53-restituted PC3 cells. Furthermore, BRCA1 protein associates to GADD153 promoter after DNA damage in the presence of p53. Additionally, we demonstrated that BRCA1 and/or p53 modulate genes involved in DNA damage and cell cycle regulation (cyclin D1, BLM, BRCA2, DDB2, p21(WAF1/CIP1), H3F3B, GADD153, GADD45A, FEN1, CCNB2), EMT (E-cadherin, ß-catenin, vimentin, fibronectin, slug, snail) and Hedgehog pathways (SHH, IHH, DHH, Gli1, PATCH1). Furthermore, xenograft studies demonstrated that BRCA1 knockdown in PC3 cells increased tumor growth and modulated these genes in vivo. CONCLUSIONS: Although BRCA1 induces GADD153 in a p53 independent manner, p53 abolished GADD153 induction in response to DNA damage. In addition, several important PCa targets are modulated by BRCA1 and p53. Altogether, these data might be important to understand the therapy response of PCa patients.

Asunto(s)

Proteína BRCA1/metabolismo , Neoplasias de la Próstata/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proteína BRCA1/genética , Ciclo Celular/genética , Línea Celular Tumoral , Daño del ADN , Erizos/genética , Erizos/metabolismo , Xenoinjertos , Humanos , Masculino , Ratones , Neoplasias de la Próstata/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factor de Transcripción CHOP/metabolismo , Transcripción Genética , Proteína p53 Supresora de Tumor/genética