Analysis of haploinsufficiency in women carrying germline mutations in the BRCA1 gene: Different mutations, different phenotypes ?

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 30-01-2015BRCA1 germline mutations are associated with significantly increased lifetime risk of developing breast and ovarian cancers. However, taking into account considerable differences in disease manifestation among mutation carriers, it is probable that various BRCA1 mutations lead to formation of distinct phenotypes and haploinsufficiency effects and that both type and location of the mutation could play an important role in cancer initiation. PARP inhibitors are currently one of the most promising agents for treatment of tumors deficient in BRCA1 or BRCA2. However, cancer patients carrying germline mutation in BRCA1/2 genes show a big variability in the responses to PARP inhibitors in clinical trials. Thus, we were interested in investigating whether and how BRCA1 mutation type influences the response of cells to PARP inhibitors We used a panel of lymphoblastoid cell lines (LCLs) derived either from heterozygous BRCA1 mutation carriers or non-carriers in order to study haploinsufficiency effects of various mutation types (truncating vs missense). LCLs carrying truncating mutations showed significantly lower BRCA1 mRNA and protein levels and higher levels of gamma-H2AX than control cells or LCLs harboring missense mutation, indicating increased spontaneous DNA damage in these cells. However, cells carrying either of the BRCA1 mutation type showed impaired RAD51 foci formation, suggesting defective repair in mutated cells and a possible mechanism of increased susceptibility of mutation carriers to cancer. Moreover, gene expression analysis revealed that LCLs carrying missense mutations showed more distinct expression profile than cells with truncating mutations when compared to controls, suggesting that different mutations may lead to distinct phenotypes and haploinsufficiency effects. Importantly, decreased expression of immune response-related genes in cells harboring missense mutation indicates possible mechanisms of breast cancer initiation in carriers of these particular BRCA1 mutations. At the present study, we are describing for the first time that cells derived from carries of missense mutations in the BRCA1 BRCT domain show higher sensitivity to PARP inhibitor olaparib than cells with truncating mutations or WTs. These results indicate that carriers of different mutation types could benefit from the treatment in a distinct way and that they could display different toxicity to the agent. Taking into account the presence of non-degraded mutated protein in cells carrying missense mutation there is a possibility that the missense mutants act in a dominant negative manner on the WT protein function. Thus, we hypothesize that there is a competition between mutated and WT BRCA1 protein in the recruitment to DNA damage sites, which could lead to an alteration the DNA repair process and decreased viability of cells carrying BRCA1 missense mutation. The mechanism of increased sensitivity of cells carrying missense mutation to olaparib could be, at least partially, also related to possible changes in olaparib-specific BRCA1 protein interactors in mutated cells. In addition, regulation of miR-222-3p and its target FOS by olaparib treatment in cells carrying missense mutation suggest a possible use of these molecules as markers of sensitivity to the treatment. In summary, our results bring new insights on how various heterozygous mutations in BRCA1 could lead to impairment of BRCA1 function and show a strong evidence about haploinsufficiency in BRCA1 mutation carriers. Moreover, our results indicate that carriers of different types of BRCA1 mutations could benefit from the treatment in a distinct way and that could show different toxicity to the PARP inhibitor olaparib.Mutaciones germinales en BRCA1 están asociadas con un incremento significativo en el riesgo a desarrollar cáncer de mama y ovario. Sin embargo, teniendo en cuenta las considerables diferencias en la manifestación de la enfermedad en portadoras de mutación, es probable que distintas mutaciones en BRCA1 den lugar a distintos fenotipos y efectos de haploinsuficiencia, y que tanto el tipo de mutación como su localización puedan jugar un papel importante en la iniciación del cáncer. Los inhibidores de PARP son actualmente uno de los agentes más prometedores para el tratamiento de tumores deficientes en BRCA1 o BRCA2. No obstante, enfermas de cáncer portadoras de una mutación germinal en los genes BRCA1/2 muestran una gran variabilidad en la respuesta a inhibidores de PARP en ensayos clínicos. Por estos motivos, nuestro interés ha radicado en investigar si el tipo de mutación en BRCA1 influye en la respuesta de las células a inhibidores de PARP y en caso afirmativo, averiguar cuál es el mecanismo responsable. Utilizamos un panel de células linfoblastoides (LCLs) derivadas bien de portadoras heterocigotas de mutación en BRCA1 o bien de no portadoras de mutación con el objetivo de estudiar los efectos de haploinsuficiencia que tienen distintos tipos de mutaciones (de proteína truncada vs de cambio de aminoácido). Las LCLs portadoras de mutaciones de proteína truncada mostraron niveles significativamente menores de ARNm y proteína BRCA1 y niveles superiores de gamma-H2AX que las células control o que las LCLs portadoras de mutación de cambio de aminoácido, lo que indica un incremento espontáneo en el daño del ADN en estas células. Sin embargo, las células portadoras tanto de un tipo como de otro de mutación, mostraron una disminución en la formación de focos de RAD51, lo que sugiere una reparación defectuosa en las células mutadas y un posible mecanismo de aumento de susceptibilidad al cáncer en portadoras de mutación. Además, los análisis de expresión génica revelaron que las LCLs portadoras de mutaciones de cambio de aminoácido mostraban perfiles de expresión más diferentes que las células con mutaciones de proteína truncada al compararlas con los controles, lo que sugiere que mutaciones diferentes podrían dar lugar a distintos fenotipos y efectos de haploinsuficiencia. De manera importante, la disminución en la expresión de genes asociados a la respuesta inmune observada en las células portadoras de mutaciones de cambio de aminoácido, indica un posible mecanismo de iniciación del cáncer de mama en portadoras de estas mutaciones concretas en BRCA1. En el presente estudio, describimos por primera vez que las células derivadas de portadoras de mutaciones de cambio de aminoácido en el dominio BRCT de BRCA1 muestran mayor sensibilidad al inhibidor de PARP olaparib que las células con mutaciones de proteína truncada o WT. Estos resultados indican que portadoras de distintos tipos de mutación podrían beneficiarse del tratamiento de distinta manera y que podrían mostrar diferente toxicidad al agente. Teniendo en cuenta la presencia de proteína mutada no degradada en las células portadoras de mutación de cambio de aminoácido, cabría la posibilidad de que los mutantes de cambio de aminoácido tengan un efecto dominante-negativo sobre la función de la proteína WT. De esta manera, planteamos la hipótesis de que existe una competición entre la proteína mutada y la WT en el reclutamiento a los sitios de daño en el ADN, lo que podría dar lugar a una alteración en el proceso de reparación del ADN y una disminución en la viabilidad de las células portadoras de mutaciones de cambio de aminoácido en BRCA1. El mecanismo de incremento en la sensibilidad a olaparib de las células portadoras de mutaciones de cambio de aminoácido podría estar asociado, al menos en parte, a posibles cambios en las proteínas específicas para olaparib que interaccionan con BRCA1 en las células mutadas. Además, la regulación del miR-222-3p y su diana FOS por el tratamiento de olaparib en células portadoras de mutaciones de cambio de aminoácido sugiere un posible uso de estas moléculas como marcadores de sensibilidad al tratamiento. En resumen, nuestros resultados aportan nuevos conocimientos sobre cómo distintas mutaciones heterocigotas en BRCA1 podrían dar lugar a defectos en la función de BRCA1 y muestran gran evidencia de haploinsuficiencia en portadoras de mutaciones en BRCA1. Además, nuestros resultados indican que portadoras de distintos tipos de mutaciones en BRCA1 podrían beneficiarse del tratamiento de distinta forma y podrían mostrar toxicidades diferentes al inhibidor de PARP olapari

DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03-1.16), p = 2.7×10-3) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8×10-3). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied

Dual Targeting of BRAF and mTOR Signaling in Melanoma Cells with Pyridinyl Imidazole Compounds

BRAF inhibitors can delay the progression of metastatic melanoma, but resistance usually emerges, leading to relapse. Drugs simultaneously targeting two or more pathways essential for cancer growth could slow or prevent the development of resistant clones. Here, we identified pyridinyl imidazole compounds SB202190, SB203580, and SB590885 as dual inhibitors of critical proliferative pathways in human melanoma cells bearing the V600E activating mutation of BRAF kinase. We found that the drugs simultaneously disrupt the BRAF V600E-driven extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activity and the mechanistic target of rapamycin complex 1 (mTORC1) signaling in melanoma cells. Pyridinyl imidazole compounds directly inhibit BRAF V600E kinase. Moreover, they interfere with the endolysosomal compartment, promoting the accumulation of large acidic vacuole-like vesicles and dynamic changes in mTOR signaling. A transient increase in mTORC1 activity is followed by the enrichment of the Ragulator complex protein p18/LAMTOR1 at contact sites of large vesicles and delocalization of mTOR from the lysosomes. The induced disruption of the endolysosomal pathway not only disrupts mTORC1 signaling, but also renders melanoma cells sensitive to endoplasmic reticulum (ER) stress. Our findings identify new activities of pharmacologically relevant small molecule compounds and provide a biological rationale for the development of anti-melanoma therapeutics based on the pyridinyl imidazole core

DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

Peer reviewe

Molecular insights into the OGG1 gene, a cancer risk modifier in BRCA1 and BRCA2 mutations carriers.

We have recently shown that rs2304277 variant in the OGG1 glycosidase gene of the Base Excision Repair pathway can increase ovarian cancer risk in BRCA1 mutation carriers. In the present study, we aimed to explore the role of this genetic variant on different genome instability hallmarks to explain its association with cancer risk.We have evaluated the effect of this polymorphism on OGG1 transcriptional regulation and its contribution to telomere shortening and DNA damage accumulation. For that, we have used a series of 89 BRCA1 and BRCA2 mutation carriers, 74 BRCAX cases, 60 non-carrier controls and 23 lymphoblastoid cell lines (LCL) derived from BRCA1 mutation carriers and non-carriers.We have identified that this SNP is associated to a significant OGG1 transcriptional down regulation independently of the BRCA mutational status and that the variant may exert a synergistic effect together with BRCA1 or BRCA2 mutations on DNA damage and telomere shortening.These results suggest that this variant, could be associated to a higher cancer risk in BRCA1 mutation carriers, due to an OGG1 transcriptional down regulation and its effect on genome instability.J.B.'s laboratory is partially funded by the Spanish Ministry of Health PI12/00070 supported by FEDER funds, and the Spanish Research Network on Rare diseases (CIBERER). C.B-B is granted by the PI12/00070. M.A.B.'s laboratory is funded with the Spanish Ministry of Science and Innovation, projects SAF2008-05384 and 2007-A-200950 (TELOMARKER), European Research Council Advanced grant GA#232854, the Korber Foundation, Fundacion Botin and Fundacion Lilly. MU is supported by the Spanish Ministry of Health PI14/00459 with FEDER funds.S

DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers.

Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03-1.16), p = 2.7 × 10(-3)) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8 × 10(-3)). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied

DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers.

Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03-1.16), p = 2.7 × 10(-3)) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8 × 10(-3)). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied

DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03-1.16), p = 2.7×10 −3) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8×10 −3). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied. Women harboring a germ-line mutation in the BRCA1 or BRCA2 genes have a high lifetime risk to develop breast and/or ovarian cancer. However, not all carriers develop cancer and high variability exists regarding age of onset of the disease and type of tumor. One of the causes of this variability lies in other genetic factors that modulate the phenotype, the so-called modifier genes. Identification of these genes might have important implications for risk assessment and decision making regarding prevention of the disease. Given that BRCA1 and BRCA2 participate in the repair of DNA double strand breaks, here we have investigated whether variations, Single Nucleotide Polymorphisms (SNPs), in genes participating in other DNA repair pathway may be associated with cancer risk in BRCA carriers. We have selected the Base Excision Repair pathway because BRCA defective cells are extremely sensitive to the inhibition of one of its components, PARP1. Thanks to a large international collaborative effort, we have been able to identify at least two SNPs that are associated with increased cancer risk in BRCA1 and BRCA2 mutation carriers respectively. These findings could have implications not only for risk assessment, but also for treatment of BRCA1/2 mutation carriers with PARP inhibitors