RESUMEN
Triple negative breast cancers (TNBCs) represent about 15-20% of all breast cancer cases and are characterized by a complex molecular heterogeneity. Some TNBCs exhibit clinical and pathological properties similar to BRCA-mutated tumors, without actually bearing a mutation in BRCA genes. This "BRCAness" phenotype may be explained by germline mutations in other genes involved in DNA repair. Although respond to chemotherapy with alkylating agents, they have a high risk of recurrence and progression. Some studies have shown the efficacy of neoadjuvant therapy in TNBC patients with DNA repair defects, but proper biomarkers of DNA repair deficiency are still needed. Here, we investigated if mutations in DNA repair genes may be correlated with anthracyclines/taxanes neoadjuvant therapy response. DNA from 19 TNBC patients undergoing neoadjuvant therapy were subjected to next generation sequencing of a panel of 24 genes in DNA repair and breast cancer predisposition. In this study, 5 of 19 patients (26%) carried a pathogenic mutation in BRCA1, PALB2, RAD51C and two patients carried a probable pathogenic missense variant. Moreover, VUS (Variants of Unknown Significance) in other genes, predicted to be deleterious by in silico tools, were detected in five patients. Germline mutations in DNA repair genes were found to be associated with the group of TNBC patients who responded to therapy. We conclude that a subgroup of TNBC patients have defects in DNA repair genes, other than BRCA1, and such patients respond favourably to neoadjuvant anthracyclines/taxanes therapy. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/genética , Enzimas Reparadoras del ADN/genética , Mutación de Línea Germinal/genética , Terapia Neoadyuvante , Recurrencia Local de Neoplasia/genética , Neoplasias de la Mama Triple Negativas/genética , Adulto , Anciano , Proteína BRCA1/genética , Estudios de Casos y Controles , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Femenino , Estudios de Seguimiento , Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Persona de Mediana Edad , Clasificación del Tumor , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , Proteínas Nucleares/genética , Pronóstico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Proteínas Supresoras de Tumor/genéticaRESUMEN
BRCA1 interacts with several proteins implicated in homologous and non homologous recombination and in mismatch repair. The aim of this study is to determine if MSH2, a well known partner of BRCA1 involved in DNA repair, may contribute to breast and ovarian cancer development and progression. To better understand the functional interaction between BRCA1 and MSH2, we studied the effect of the deletion of MSH2 gene on BRCA1-induced genome instability in yeast. Preliminary results in yeast indicated that MSH2 and BRCA1 may interact to modulate homologous recombination (HR). We also carried out a genetic and epigenetic profiling of MSH2 gene by mutational analysis and promoter methylation evaluation in 9 breast and 2 ovarian tumors from carriers of BRCA1 unknown significance variants (VUS). 2/2 ovarian and 2/9 breast tumors carried MSH2 somatic mutations possible pathogenics (4/11, 36%): a missense mutation in exon 3 (p.G162R), a duplication of exon 1 and a deletion of exon 2. In addition, two germline synonymous variants in exon 11 were identified. None of the tumors showed promoter methylation. In conclusion, a surprisingly high frequency of MSH2 gene mutations has been found in tumor tissues from BRCA1 VUS carrier patients. This result supports the indication deriving from the yeast model that BRCA1 driven tumorigenesis may be modulated by MSH2.
Asunto(s)
Proteína BRCA1/genética , Neoplasias de la Mama/genética , Carcinogénesis/genética , Carcinoma/genética , Proteína 2 Homóloga a MutS/genética , Proteína BRCA1/metabolismo , Metilación de ADN , Exones , Femenino , Recombinación Homóloga , Humanos , Proteína 2 Homóloga a MutS/metabolismo , Tasa de Mutación , Polimorfismo Genético , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
M1775R and A1789T are two missense variants located within the BRCT domains of BRCA1 gene. The M1775R is a known deleterious variant, while the A1789T is an unclassified variant that has been analyzed and classified as probably deleterious for the first time by our group. In a previous study, we described the expression profile of HeLa G1 cells transfected with the two variants and we found that they altered molecular mechanisms critical for cell proliferation and genome integrity. Considering that the mutations in the BRCA1 C terminus (BRCT) domains are associated to a phenotype with an altered ability in the DNA double-strand break repair, we chose three of the genes previously identified, EEF1E1, MRE11A, and OBFC2B, to be tested for an homologous recombination (HR) in vitro assay. For our purpose, we performed a gene expression knockdown by siRNA transfection in HeLa cells, containing an integrated recombination substrate (hprtDRGFP), for each of the target genes included BRCA1. The knockdown of BRCA1, OBFC2B, MRE11A, and EEF1E1 reduces the HR rate, respectively, of 97.6, 28.6, 41.8, and 42.3 % compared to cells transfected with a scrambled negative control duplex and all these differences are statistically significant (P < 0.05; Kruskal-Wallis test). Finally, we analyzed the effect of target gene depletion both on HR that on cell survival after DNA-damage induction by ionizing radiation. The clonogenic assay showed that the down-regulation of the target genes reduced the cell survival, but the effect on the HR, is not evident. Only the BRCA1-siRNA confirmed the inhibition effect on HR. Overall these results confirmed the involvement of MRE11A in the HR pathway and identified two new genes, OBFC2B and EEF1E1, which according to these data and the knowledge obtained from literature, might be involved in BRCA1-pathway.
Asunto(s)
Proteínas de Unión al ADN/genética , Genes BRCA1 , Proteínas Mitocondriales/genética , Factores de Elongación de Péptidos/genética , Reparación del ADN por Recombinación , Proteínas Supresoras de Tumor/genética , Supervivencia Celular/efectos de la radiación , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , Técnicas de Silenciamiento del Gen , Estudios de Asociación Genética , Células HeLa , Humanos , Proteína Homóloga de MRE11 , Proteínas Mitocondriales/metabolismo , Factores de Elongación de Péptidos/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Cytosolic 5'-nucleotidase II is a widespread IMP hydrolyzing enzyme, essential for cell vitality, whose role in nucleotide metabolism and cell function is still to be exactly determined. Cytosolic 5'-nucleotidase overexpression and silencing have both been demonstrated to be toxic for mammalian cultured cells. In order to ascertain the effect of enzyme expression on a well-known eukaryote simple model, we expressed cytosolic 5'-nucleotidase II in Saccharomyces cerevisiae, which normally hydrolyzes IMP through the action of a nucleotidase with distinct functional and structural features. Heterologous expression was successful. The yeast cells harbouring cytosolic 5'-nucleotidase II displayed a shorter duplication time and a significant modification of purine and pyrimidine derivatives concentration as compared with the control strain. Furthermore the capacity of homologous recombination in the presence of mutagenic compounds of yeast expressing cytosolic 5'-nucleotidase II was markedly impaired.
Asunto(s)
5'-Nucleotidasa/metabolismo , Bovinos/metabolismo , Citosol/enzimología , Recombinación Homóloga/fisiología , Nucleótidos/análisis , Saccharomyces cerevisiae/crecimiento & desarrollo , Análisis de Varianza , Animales , Western Blotting , División Celular/fisiología , Cartilla de ADN/genética , Electroforesis en Gel de Poliacrilamida , Saccharomyces cerevisiae/metabolismo , TemperaturaRESUMEN
The tumour-suppressor gene BRCA2 has been demonstrated to be involved in maintenance of genome integrity by affecting DNA double-strand break repair and homologous recombination. Protein-truncating mutations in BRCA2 predispose women to early onset breast and ovarian cancers and account for 15-30% of familial breast cancer risk. In contrast, the human cancer risk due to missense mutations, intronic variants, and in-frame deletions and insertions in the BRCA2 gene, called unclassified variants, has not been determined. Here, we want to define if the yeast Saccharomyces cerevisiae is a good model to study the role of BRCA2 in DNA recombination and repair and to characterise the unclassified BRCA2 missense variants. Therefore, we expressed the wild-type BRCA2 in yeast and determined the effect of BRCA2 on yeast homologous recombination, methyl methanesulphonate (MMS)-induced Rad51 and Rad52 foci and MMS sensitivity. The expression of BRCA2 induces a high increase in both intra- and inter-recombination events and confers a higher MMS resistance as compared with the negative control. This may suggest that BRCA2 gets involved in DNA repair pathways in yeast. Moreover, the expression of BRCA2 did not affect the number of cells carrying Rad51 or Rad52 nuclear foci. Finally, we aimed to investigate if yeast could be reliable system to set up a functional assay to distinguish a mutated protein from a neutral polymorphism. Therefore, we have expressed two neutral (M1915T and A2951T) and one pathogenic variant (G2748D) in yeast and checked the effect on recombination. The neutral M1915T variant increased intra-chromosomal recombination by almost 2-fold and the other neutral A2975T variant increased intra-chromosomal recombination 2.5-fold as compared with the control. On the other end, the pathogenic variant G2748D did not increase intra- and inter-chromosomal recombination in yeast and, consequently, confers a phenotype very different from the wild-type BRCA2. Moreover, we have also evaluated whether the expression of the selected unclassified variants affects homologous recombination in yeast. Results indicated that the expression of the selected BRCA2 variants differentially affects yeast recombination suggesting that yeast could be a very promising genetic system to characterise BRCA2 missense variants.
Asunto(s)
Proteína BRCA2/genética , Núcleo Celular/genética , Daño del ADN/efectos de los fármacos , Recombinación Homóloga , Saccharomyces cerevisiae/genética , Proteína BRCA2/metabolismo , Núcleo Celular/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Metilmetanosulfonato/farmacología , Mutación Missense , Fenotipo , Plásmidos , Polimorfismo Genético , Regiones Promotoras Genéticas , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
OBJECTIVE: Breast cancer is the most common female cancer in Morocco. About 5 to 10% are due to hereditary predisposition and mutations in BRCA1 and BRCA2 genes are responsible for an important proportion of high-risk breast/ovarian cancer families. The relevance of BRCA1/2 mutations in the Moroccan population was not studied. The main objective of this study is to investigate the spectrum of BRCA1 and BRCA2 germline mutations in early onset and familial breast/ovarian cancer among Moroccan women. METHODS: We screened the entire coding sequences and intron/exon boundaries of BRCA1 and BRCA2 genes in 40 patients by direct sequencing. RESULTS: Nine pathogenic mutations were detected in ten unrelated families, five deleterious mutations in BRCA1 gene and four mutations in BRCA2 gene. Four novel mutations were found: one in BRCA1 (c.2805delA/2924delA) and three in BRCA2 (c.3381delT/3609delT; c.7110delA/7338delA and c.7235insG/7463insG). We also identified 51 distinct polymorphisms and unclassified variants (three described for the first time). CONCLUSIONS: Our data suggest that BRCA1 and BRCA2 mutations are responsible for a significant proportion of familial breast cancer in Moroccan patients. Therefore full BRCA1/2 screening should be offered to patients with a family history of breast/ovarian cancer.
Asunto(s)
Neoplasias de la Mama/genética , Genes BRCA1 , Genes BRCA2 , Mutación de Línea Germinal , Neoplasias Ováricas/genética , Adulto , Secuencia de Bases , Neoplasias de la Mama Masculina/genética , Análisis Mutacional de ADN , Exones , Salud de la Familia , Femenino , Eliminación de Gen , Humanos , Intrones , Masculino , Datos de Secuencia Molecular , Marruecos , Linaje , Polimorfismo GenéticoRESUMEN
IMP preferring cytosolic 5'-nucleotidase II (cN-II) is a widespread enzyme whose amino acid sequence is highly conserved among vertebrates. Fluctuations of its activity have been reported in some pathological conditions and its mRNA levels have been proposed as a prognostic factor for poor outcome in patients with adult acute myeloid leukemia. As a member of the oxypurine cycle, cN-II is involved in the regulation of intracellular concentration of 5'-inosine monophosphate (IMP), 5'-guanosine monophosphate (GMP), and also 5-phosphoribose 1-pyrophosphate (PRPP) and is therefore involved in the regulation of purine and pyrimidine de novo and salvage synthesis. In addition, several studies demonstrated the involvement of cN-II in pro-drug metabolism. Notwithstanding some publications indicating that cN-II is essential for the survival of several cell types, its role in cell metabolism remains uncertain. To address this issue, we built two eucaryotic cellular models characterized by different cN-II expression levels: a constitutive cN-II knockdown in the astrocytoma cell line (ADF) by short hairpin RNA (shRNA) strategy and a cN-II expression in the diploid strain RS112 of Saccharomyces cerevisiae. Preliminary results suggest that cN-II is essential for cell viability, probably because it is directly involved in the regulation of nucleotide pools. These two experimental approaches could be very useful for the design of a personalized chemotherapy.
Asunto(s)
5'-Nucleotidasa/metabolismo , 5'-Nucleotidasa/genética , Astrocitoma/enzimología , Astrocitoma/genética , Línea Celular Tumoral , Supervivencia Celular , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recombinación Genética/genética , Transducción GenéticaRESUMEN
By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the ß2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of ß2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the ß2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the ß2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.
Asunto(s)
Reparación del ADN/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Recombinasa Rad51/metabolismo , Recombinación Genética/genética , Proteínas de Saccharomyces cerevisiae/genética , Dominio Catalítico/genética , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Daño del ADN , Células HeLa/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/metabolismo , TransfecciónRESUMEN
To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR.
Asunto(s)
Recombinación Genética , Saccharomyces cerevisiae/genética , Reparación del ADN , Biblioteca de Genes , Genes ras , Humanos , Proteínas de la Membrana/genética , Metilmetanosulfonato/farmacología , Proteínas del Tejido Nervioso/genética , Complejo de la Endopetidasa Proteasomal/genética , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/fisiología , Proteínas Represoras/genética , Proteínas Ribosómicas/genéticaRESUMEN
The TBX5 transcription factor is required for normal cardiogenesis, and human TBX5 mutations cause congenital heart defects. Previous studies have shown that TBX5 can localize to cellular nuclei during embryogenesis and have suggested that altered nuclear localization may contribute to disease pathogenesis. Current analyses suggest that TBX5 nuclear localization is not uniform during organogenesis. To determine the biochemical mechanisms underlying TBX5 nuclear import, we performed site-directed mutagenesis of human TBX5. We identified two distinct nuclear localization signals in TBX5, one monopartite and one bipartite. While each is insufficient to promote complete TBX5 nuclear localization, they act cooperatively to do so. These sequences are evolutionarily conserved and have cognates in other T-box gene family members.
Asunto(s)
Núcleo Celular/metabolismo , Mutación , Proteínas de Dominio T Box/biosíntesis , Transporte Activo de Núcleo Celular , Animales , Células COS , ADN Complementario/metabolismo , Exones , Cardiopatías Congénitas/patología , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Modelos Moleculares , Mutagénesis , Transducción de Señal , TransfecciónRESUMEN
Reports related some polymorphisms of the 5,10-methylenetetrahydrofolate reductase (MTHFR) to folate-dependent neural tube defects. In view of the common origin of the cells involved both in neural tube closure and heart septation, we analyzed the MTHFR C677T and A1298C polymorphisms in mothers of children with conotruncal heart defect (CD) and in their offspring to evaluate the association between the MTHFR genotype and the risk of CD. We genotyped 103 Italian mothers with CD offspring, 200 control mothers, 103 affected children and their fathers by restriction fragment length polymorphism analysis. No increased risk was observed for the prevalence of the 677TT genotype by itself in affected children and in their mothers. The combined maternal 677TT/1298AA and 677CC/1298CC genotypes have odds ratio of 1.73 and 1.85, respectively. The prevalence of 1298CC genotype in the affected children gives odds ratio of 1.90, that becomes 2.31 for the 677CC/1298CC genotype. However, none of the odds ratios was statistically significant. We observed a higher frequency of the 677T allele in Italy than in other European countries. No association has been demonstrated between the 677TT MTHFR genotype and CD.