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In this paper, the mesophilic Biochemical Methane Potential of several fabrics was assessed at different Total Solid concentrations (1-4%TS). Physico-chemical techniques were applied to explore the arising structural changes on fibers during the anaerobic digestion process. Additionally, the modified Gompertz model was used to assess and compare the AD performance of the fabrics. In cellulose-based fibers the production of biogas was enhanced thanks to the easy solubilization of acetate, which is generated upon partial breakage of cellulose bonds. The crystallinity of vegetal fibers decreased significantly from day 19. The highest methane yields were attained for silk and wool fabrics at the lowest TS concentrations. Conformational changes in fibroin and keratin were detected. The highest degrees of degradation were observed in solid samples with lower solid concentrations. Accordingly, the maximum methane yields were reported in the reactors operating with lower TS.
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Celulose , Metano , Animais , Anaerobiose , Reatores Biológicos , BiocombustíveisRESUMO
Collagen-based polymers and their blends have attracted considerable interest for new materials development due to their unique combination of biocompatibility, physical and mechanical properties and durability. Leather, a modified natural biopolymer made from animal rawhide and the first synthetic collagen-based polymer known since the dawn of civilization, combines all these features. Rawhide is transformed into leather by tanning, a process in which the collagen is cross-linked with different agents to make it stronger and more durable and to prevent its decay. Research on the development of environmentally friendly procedures and sustainable materials with higher efficiency and lower costs is a rapidly growing field, and leather industry is not an exemption. Chrome-tanned and vegetable-tanned (chromium-free) shavings from the leather industry present a high content of organic matter, yet they are considered recalcitrant waste to be degraded by microbiological processes like anaerobic digestion (AD), a solid technology to treat organic waste in a circular economy framework. In this technology however, the solubilisation of organic solid substrates is a significant challenge to improving the efficiency of the process. In this context, we have investigated the process of microbial decomposition of leather wastes from the tannery industry to search for the conditions that produce optimal solubilisation of organic matter. Chrome-tanned and chromium-free leather shavings were pre-treated and anaerobically digested under different temperature ranges (thermophilic-55 °C-, intermediate-42 °C- and mesophilic-35 °C) to evaluate the effect on the solubilisation of the organic matter of the wastes. The results showed that the presence of chromium significantly inhibited the solubilization (up to 60%) in the mesophilic and intermediate ranges; this is the fastest and most efficient solubilization reached under thermophilic conditions using the chromium-free leather shaving as substrates. The most suitable temperature for the solubilization was the thermophilic regime (55 °C) for both chromium-free and chrome-tanned shavings. No significant differences were observed in the thermophilic anaerobic digestion of chromium-free shavings when a pre-treatment was applied, since the solubilisation was already high without pre-treatment. However, the pre-treatments significantly improved the solubilisation in the mesophilic and intermediate configurations; the former pre-treatment was better suited in terms of performance and cost-effectiveness compared to the thermophilic range. Thus, the solubilisation of chromium-free tannery solid wastes can be significantly improved by applying appropriate pre-treatments at lower temperature ranges; this is of utter importance when optimizing anaerobic processes of recalcitrant organic wastes, with the added benefit of substantial energy savings in the scaling up of the process in an optimised circular economy scenario.
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Resíduos Industriais , Curtume , Animais , Cromo/química , Temperatura , Anaerobiose , ColágenoRESUMO
Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas with poor prognosis, developing either sporadically or in persons with neurofibromatosis type 1 (NF1). Loss of CDKN2A/B is an important early event in MPNST progression. However, many reported MPNSTs exhibit partial or no inactivation of CDKN2A/B, raising the question of whether there is more than one molecular path for MPNST initiation. We present here a comprehensive genomic analysis of MPNST cell lines and tumors to explore in depth the status of CDKN2A. After accounting for CDKN2A deletions and point mutations, we uncovered a previously unnoticed high frequency of chromosomal translocations involving CDKN2A in both MPNST cell lines and primary tumors. Most identified translocation breakpoints were validated by PCR amplification and Sanger sequencing. Many breakpoints clustered in an intronic 500 bp hotspot region adjacent to CDKN2A exon 2. We demonstrate the bi-allelic inactivation of CDKN2A in all tumors (n = 15) and cell lines (n = 8) analyzed, supporting a single molecular path for MPNST initiation in both sporadic and NF1-related MPNSTs. This general CDKN2A inactivation in MPNSTs has implications for MPNST diagnostics and treatment. Our findings might be relevant for other tumor types with high frequencies of CDKN2A inactivation.
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Carcinogênese/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Neurofibromatose 1/genética , Neurofibrossarcoma/genética , Polimorfismo de Nucleotídeo Único , Sarcoma/genética , Translocação Genética , Sequência de Bases , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Cromossomos Humanos Par 9 , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Éxons , Genoma Humano , Humanos , Neurofibromatose 1/complicações , Neurofibromatose 1/metabolismo , Neurofibromatose 1/patologia , Neurofibrossarcoma/etiologia , Neurofibrossarcoma/metabolismo , Neurofibrossarcoma/patologia , Sarcoma/etiologia , Sarcoma/metabolismo , Sarcoma/patologia , Células de Schwann/metabolismo , Células de Schwann/patologia , Sequenciamento Completo do GenomaRESUMO
PURPOSE: Autism spectrum disorders are associated with defects in social response and communication that often occur in the context of intellectual disability. Rett syndrome is one example in which epilepsy, motor impairment, and motor disturbance may co-occur. Mutations in histone demethylases are known to occur in several of these syndromes. Herein, we aimed to identify whether mutations in the candidate histone demethylase JMJD1C (jumonji domain containing 1C) are implicated in these disorders. METHODS: We performed the mutational and functional analysis of JMJD1C in 215 cases of autism spectrum disorders, intellectual disability, and Rett syndrome without a known genetic defect. RESULTS: We found seven JMJD1C variants that were not present in any control sample (~ 6,000) and caused an amino acid change involving a different functional group. From these, two de novo JMJD1C germline mutations were identified in a case of Rett syndrome and in a patient with intellectual disability. The functional study of the JMJD1C mutant Rett syndrome patient demonstrated that the altered protein had abnormal subcellular localization, diminished activity to demethylate the DNA damage-response protein MDC1, and reduced binding to MECP2. We confirmed that JMJD1C protein is widely expressed in brain regions and that its depletion compromises dendritic activity. CONCLUSIONS: Our findings indicate that mutations in JMJD1C contribute to the development of Rett syndrome and intellectual disability.Genet Med 18 1, 378-385.
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Deficiência Intelectual/genética , Histona Desmetilases com o Domínio Jumonji/genética , Mutação , Oxirredutases N-Desmetilantes/genética , Síndrome de Rett/genética , Adulto , Motivos de Aminoácidos , Sequência de Aminoácidos , Transtorno do Espectro Autista/diagnóstico , Transtorno do Espectro Autista/genética , Encéfalo/metabolismo , Encéfalo/patologia , Sequência Conservada , Análise Mutacional de DNA , Feminino , Expressão Gênica , Ordem dos Genes , Estudos de Associação Genética , Loci Gênicos , Humanos , Deficiência Intelectual/diagnóstico , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Neurônios/metabolismo , Oxirredutases N-Desmetilantes/química , Oxirredutases N-Desmetilantes/metabolismo , Matrizes de Pontuação de Posição Específica , Conformação Proteica , Transporte Proteico , Síndrome de Rett/diagnósticoRESUMO
Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft-tissue sarcomas with a poor survival rate, presenting either sporadically or in the context of neurofibromatosis type 1 (NF1). The histological diagnosis of MPNSTs can be challenging, with different tumors exhibiting great histological and marker expression overlap. This heterogeneity could be partly responsible for the observed disparity in treatment response due to the inherent diversity of the preclinical models used. For several years, our group has been generating a large patient-derived orthotopic xenograft (PDOX) MPNST platform for identifying new precision medicine treatments. Herein, we describe the expansion of this platform using six primary tumors clinically diagnosed as MPNSTs, from which we obtained six additional PDOX mouse models and three cell lines, thus generating three pairs of in vitro-in vivo models. We extensively characterized these tumors and derived preclinical models, including genomic, epigenomic, and histological analyses. Tumors were reclassified after these analyses: three remained as MPNSTs (two being classic MPNSTs), one was a melanoma, another was a neurotrophic tyrosine receptor kinase (NTRK)-rearranged spindle cell neoplasm, and, finally, the last was an unclassifiable tumor bearing neurofibromin-2 (NF2) inactivation, a neuroblastoma RAS viral oncogene homolog (NRAS) oncogenic mutation, and a SWI/SNF-related matrix-associated actin-dependent regulator of chromatin (SMARCA4) heterozygous truncated variant. New cell lines and PDOXs faithfully recapitulated histology, marker expression, and genomic characteristics of the primary tumors. The diversity in tumor identity and their specific associated genomic alterations impacted treatment responses obtained when we used the new cell lines for testing compounds against known altered pathways in MPNSTs. In summary, we present here an extension of our MPNST precision medicine platform, with new PDOXs and cell lines, including tumor entities confounded as MPNSTs in a real clinical scenario. This platform may constitute a useful tool for obtaining correct preclinical information to guide MPNST clinical trials.
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Neoplasias de Bainha Neural , Neurofibrossarcoma , Humanos , Camundongos , Animais , Neurofibrossarcoma/genética , Neoplasias de Bainha Neural/genética , Neoplasias de Bainha Neural/patologia , Medicina de Precisão , Xenoenxertos , Linhagem Celular , DNA Helicases , Proteínas Nucleares , Fatores de TranscriçãoRESUMO
The local waste co-digestion is an interesting option to tackle in reduced and isolated areas like the islands. The islands have limited territory and scarce fuel production. Moreover, organic waste can create serious environmental problems in soil, water and air. Anaerobic co-digestion (AcoD) is a technology fulfilling the concept of waste-to-energy (WtE) based on local resources. The valorisation of organic waste through AcoD on an island would prevent environmental impacts, while being a source of renewable energy. In this study, cow manure (outdoor and indoor), pig slurry, bird manure, kitchen waste, sewage sludge and oily lacteous waste produced on Island Terceira (Portugal) were tested in mesophilic -35 °C- Biochemical Methane Potential (BMP) co-digestion assays. The goals were to analyse the recalcitrant and high potential produced waste and to estimate the energetic supply source on the island. The cow manure and pig slurry were used as inocula and specific methanogenic activities (SMAs) were carried out. The results showed that both substrates have a significant methanogenic activity-SMA 0.11 g-COD/(g-VSS.d) and 0.085 g-COD/(g-VSS.d), respectively. All the studied combinations were feasible in AcoD, showing TS removals in the range of 19-37%; COD removals in the range 67-78% and specific methane yields from 0.14 to 0.22 L/gCOD removed, but some differences were found. The modified Gompertz model fitted the AcoD assays (R2 0.982-0.998). The maximum biogas production rate, Rmax. was highest in the AcoD of Cow+Pig+Oily and in the Cow+Pig+Sludge with 0.017 and 0.014 L/g-VSadded.day, respectively, and the lowest in Cow+Pig+Bird with 0.010 L/g-VSadded. In our AcoD studies, the bird manure limited the performance of the process, since it was recalcitrant to anaerobic degradation. On the other hand, the oily lacteous waste showed a great potential in the anaerobic digestion. The estimated biogas production, from the best-studied condition, could cover the 11.4% of the energy supply of the inhabitants. These preliminary results would prevent the environmental impact of organic waste on the island and promote the use of local waste in a circular economy scenario.
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Neurofibromas are benign peripheral nervous system tumors associated with neurofibromatosis type 1, which originate from NF1(-/-) Schwann cell precursors. We describe a protocol to generate neurofibromaspheres by differentiating NF1(-/-) Schwann cells from induced pluripotent stem cells and combining them with neurofibroma primary fibroblasts. We also describe the development of neurofibroma-like tumors when neurofibromaspheres are engrafted in the sciatic nerve of nude mice. This model constitutes a versatile platform for drug screening and the study of neurofibroma biology. For complete details on the use and execution of this protocol, please refer to Mazuelas et al. (2022).1.
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Malignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas of the peripheral nervous system that develop either sporadically or in the context of neurofibromatosis type 1 (NF1). MPNST diagnosis can be challenging and treatment outcomes are poor. We present here a resource consisting of the genomic characterization of 9 widely used human MPNST cell lines for their use in translational research. NF1-related cell lines recapitulated primary MPNST copy number profiles, exhibited NF1, CDKN2A, and SUZ12/EED tumor suppressor gene (TSG) inactivation, and presented no gain-of-function mutations. In contrast, sporadic cell lines collectively displayed different TSG inactivation patterns and presented kinase-activating mutations, fusion genes, altered mutational frequencies and COSMIC signatures, and different methylome-based classifications. Cell lines re-classified as melanomas and other sarcomas exhibited a different drug-treatment response. Deep genomic analysis, methylome-based classification, and cell-identity marker expression, challenged the identity of common MPNST cell lines, opening an opportunity to revise MPNST differential diagnosis.
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PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive soft-tissue sarcomas that lack effective treatments, underscoring the urgent need to uncover novel mediators of MPNST pathogenesis that may serve as potential therapeutic targets. Tumor angiogenesis is considered a critical event in MPNST transformation and progression. Here, we have investigated whether endoglin (ENG), a TGFß coreceptor with a crucial role in angiogenesis, could be a novel therapeutic target in MPNSTs. EXPERIMENTAL DESIGN: ENG expression was evaluated in human peripheral nerve sheath tumor tissues and plasma samples. Effects of tumor cell-specific ENG expression on gene expression, signaling pathway activation and in vivo MPNST growth and metastasis, were investigated. The efficacy of ENG targeting in monotherapy or in combination with MEK inhibition was analyzed in xenograft models. RESULTS: ENG expression was found to be upregulated in both human MPNST tumor tissues and plasma-circulating small extracellular vesicles. We demonstrated that ENG modulates Smad1/5 and MAPK/ERK pathway activation and pro-angiogenic and pro-metastatic gene expression in MPNST cells and plays an active role in tumor growth and metastasis in vivo. Targeting with ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST growth and metastasis in xenograft models by reducing tumor cell proliferation and angiogenesis. Moreover, combination of anti-ENG therapy with MEK inhibition effectively reduced tumor cell growth and angiogenesis. CONCLUSIONS: Our data unveil a tumor-promoting function of ENG in MPNSTs and support the use of this protein as a novel biomarker and a promising therapeutic target for this disease.
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Neoplasias de Bainha Neural , Neurofibrossarcoma , Humanos , Biomarcadores , Linhagem Celular Tumoral , Endoglina/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias de Bainha Neural/tratamento farmacológico , Neoplasias de Bainha Neural/genética , Neoplasias de Bainha Neural/metabolismo , Transdução de SinaisRESUMO
Comprehensive genetic testing of the breast cancer susceptibility genes BRCA1 and BRCA2 identified approximately 16% of variants of unknown significance (VUS), a significant proportion of which could affect the correct splicing of the genes. Our aim is to establish a workflow for classifying VUS in these complex genes, the first stage of which is splicing analysis. We used a combined approach consisting of five in silico splicing prediction programs and RT-PCR analysis for a set of 26 variants not previously studied at the mRNA level and six variants that had already been studied, four of which were used as positive controls as they were found to affect the splicing of these genes and the other two were used as negative controls. We identified a splicing defect in 8 of the 26 newly studied variants and ruled out splicing alteration in the remaining 18 variants. The results for the four positive and the two negative control variants were consistent with results presented in the literature. Our results strongly suggest that the combination of RNA analysis and in silico programs is an important step towards the classification of VUS. The results revealed a very high correlation between experimental data and in silico programs when using tools for predicting acceptor/donor sites but a lower correlation in the case of tools for identifying ESE elements.
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Proteína BRCA1/genética , Proteína BRCA2/genética , Síndrome Hereditária de Câncer de Mama e Ovário/genética , RNA Mensageiro/genética , Processamento Alternativo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Sequência de Bases , Simulação por Computador , Feminino , Predisposição Genética para Doença , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de RNARESUMO
BACKGROUND: Genes that, when mutated, cause Fanconi anemia or greatly increase breast cancer risk encode for proteins that converge on a homology-directed DNA damage repair process. Mutations in the SLX4 gene, which encodes for a scaffold protein involved in the repair of interstrand cross-links, have recently been identified in unclassified Fanconi anemia patients. A mutation analysis of SLX4 in German or Byelorussian familial cases of breast cancer without detected mutations in BRCA1 or BRCA2 has been completed, with globally negative results. METHODS: The genomic region of SLX4, comprising all exons and exon-intron boundaries, was sequenced in 94 Spanish familial breast cancer cases that match a criterion indicating the potential presence of a highly-penetrant germline mutation, following exclusion of BRCA1 or BRCA2 mutations. RESULTS: This mutational analysis revealed extensive genetic variation of SLX4, with 21 novel single nucleotide variants; however, none could be linked to a clear alteration of the protein function. Nonetheless, genotyping 10 variants (nine novel, all missense amino acid changes) in a set of controls (138 women and 146 men) did not detect seven of them. CONCLUSIONS: Overall, while the results of this study do not identify clearly pathogenic mutations of SLX4 contributing to breast cancer risk, further genetic analysis, combined with functional assays of the identified rare variants, may be warranted to conclusively assess the potential link with the disease.
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Neoplasias da Mama/genética , Genes BRCA1 , Genes BRCA2 , Mutação de Sentido Incorreto/genética , Recombinases/genética , Análise Mutacional de DNA , Éxons/genética , Família , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa/genética , Humanos , Íntrons/genética , MasculinoRESUMO
Malignant peripheral nerve sheath tumors (MPNST) are soft-tissue sarcomas that are the leading cause of mortality in patients with Neurofibromatosis type 1 (NF1). Single chemotherapeutic agents have shown response rates ranging from 18% to 44% in clinical trials, so there is still a high medical need to identify chemotherapeutic combination treatments that improve clinical prognosis and outcome. We screened a collection of compounds from the NCATS Mechanism Interrogation PlatE (MIPE) library in three MPNST cell lines, using cell viability and apoptosis assays. We then tested whether compounds that were active as single agents were synergistic when screened as pairwise combinations. Synergistic combinations in vitro were further evaluated in patient-derived orthotopic xenograft/orthoxenograft (PDOX) athymic models engrafted with primary MPNST matching with their paired primary-derived cell line where synergism was observed. The high-throughput screening identified 21 synergistic combinations, from which four exhibited potent synergies in a broad panel of MPNST cell lines. One of the combinations, MK-1775 with Doxorubicin, significantly reduced tumor growth in a sporadic PDOX model (MPNST-SP-01; sevenfold) and in an NF1-PDOX model (MPNST-NF1-09; fourfold) and presented greater effects in TP53 mutated MPNST cell lines. The other three combinations, all involving Panobinostat (combined with NVP-BGT226, Torin 2, or Carfilzomib), did not reduce the tumor volume in vivo at noncytotoxic doses. Our results support the utility of our screening platform of in vitro and in vivo models to explore new therapeutic approaches for MPNSTs and identified that combination MK-1775 with Doxorubicin could be a good pharmacologic option for the treatment of these tumors.
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Neoplasias de Bainha Neural , Neurofibromatose 1 , Neurofibrossarcoma , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias de Bainha Neural/tratamento farmacológico , Neoplasias de Bainha Neural/genética , Neurofibromatose 1/complicações , Neurofibromatose 1/patologia , Neurofibromatose 1/terapiaRESUMO
Plexiform neurofibromas (pNFs) are developmental tumors that appear in neurofibromatosis type 1 individuals, constituting a major source of morbidity and potentially transforming into a highly metastatic sarcoma (MPNST). pNFs arise after NF1 inactivation in a cell of the neural crest (NC)-Schwann cell (SC) lineage. Here, we develop an iPSC-based NC-SC in vitro differentiation system and construct a lineage expression roadmap for the analysis of different 2D and 3D NF models. The best model consists of generating heterotypic spheroids (neurofibromaspheres) composed of iPSC-derived differentiating NF1(-/-) SCs and NF1(+/-) pNF-derived fibroblasts (Fbs). Neurofibromaspheres form by maintaining highly proliferative NF1(-/-) cells committed to the NC-SC axis due to SC-SC and SC-Fb interactions, resulting in SC linage cells at different maturation points. Upon engraftment on the mouse sciatic nerve, neurofibromaspheres consistently generate human NF-like tumors. Analysis of expression roadmap genes in human pNF single-cell RNA-seq data uncovers the presence of SC subpopulations at distinct differentiation states.
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Células-Tronco Pluripotentes Induzidas/patologia , Neurofibroma Plexiforme/patologia , Células de Schwann/patologia , Adolescente , Adulto , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Criança , Feminino , Humanos , Masculino , Mesoderma/patologia , Camundongos , Pessoa de Meia-Idade , Modelos Biológicos , Crista Neural/patologia , Nervo Isquiático/patologia , Esferoides Celulares/patologia , Adulto JovemRESUMO
Here we analyze the genetic and molecular basis responsible for a very benign phenotype observed in an NF1 patient. Quantification of cells carrying the NF1 mutation in different samples derived from the three embryonic layers revealed mosaicism. Furthermore, the construction of a minigene with patient's mutation (c.3198 - 314G>A) confirmed its benign nature due to the leakiness of the splicing mechanism that generated a proportion of correctly spliced transcripts. Hence, we concluded that the mild phenotype observed in this patient is the result of the presence of mosaicism together with the benign nature of a leaky NF1-splice mutation. Finally, with the aim of developing a personalized therapeutic approach for this patient, we demonstrated correction of the splicing defect by using specific antisense morpholino oligomers. Our results provide an example of the molecular complexity behind disease phenotypes and highlight the importance of using comprehensive genetic approaches to better assess phenotype-genotype correlations.
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Mosaicismo , Neurofibromatose 1/genética , Neurofibromatose 1/patologia , Neurofibromina 1/genética , Adulto , Feminino , Estudos de Associação Genética , Genótipo , Humanos , Mutação/genética , Oligorribonucleotídeos Antissenso , Fenótipo , Medicina de Precisão , Isoformas de Proteínas/genética , Splicing de RNA/genéticaRESUMO
Dermal neurofibromas (dNFs) are benign tumors of the peripheral nervous system typically associated with Neurofibromatosis type 1 (NF1) patients. Genes controlling the integrity of the DNA are likely to influence the number of neurofibromas developed because dNFs are caused by somatic mutational inactivation of the NF1 gene, frequently evidenced by loss of heterozygosity (LOH). We performed a comprehensive analysis of the prevalence and mechanisms of LOH in dNFs. Our study included 518 dNFs from 113 patients. LOH was detected in 25% of the dNFs (N = 129). The most frequent mechanism causing LOH was mitotic recombination, which was observed in 62% of LOH-tumors (N = 80), and which does not reduce the number of NF1 gene copies. All events were generated by a single crossover located between the centromere and the NF1 gene, resulting in isodisomy of 17q. LOH due to the loss of the NF1 gene accounted for a 38% of dNFs with LOH (N = 49), with deletions ranging in size from â¼80 kb to â¼8 Mb within 17q. In one tumor we identified the first example of a neurofibroma-associated second-hit type-2 NF1 deletion. Analysis of the prevalence of mechanisms causing LOH in dNFs in individual patients (possibly under genetic control) will elucidate whether there exist interindividual variation.
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Perda de Heterozigosidade/genética , Neurofibroma/genética , Neurofibromatose 1/genética , Técnicas de Cultura de Células , Pontos de Quebra do Cromossomo , Deleção Cromossômica , Variações do Número de Cópias de DNA/genética , Frequência do Gene/genética , HumanosRESUMO
INTRODUCTION: Proteins encoded by Fanconi anemia (FA) and/or breast cancer (BrCa) susceptibility genes cooperate in a common DNA damage repair signaling pathway. To gain deeper insight into this pathway and its influence on cancer risk, we searched for novel components through protein physical interaction screens. METHODS: Protein physical interactions were screened using the yeast two-hybrid system. Co-affinity purifications and endogenous co-immunoprecipitation assays were performed to corroborate interactions. Biochemical and functional assays in human, mouse and Caenorhabditis elegans models were carried out to characterize pathway components. Thirteen FANCD2-monoubiquitinylation-positive FA cell lines excluded for genetic defects in the downstream pathway components and 300 familial BrCa patients negative for BRCA1/2 mutations were analyzed for genetic mutations. Common genetic variants were genotyped in 9,573 BRCA1/2 mutation carriers for associations with BrCa risk. RESULTS: A previously identified co-purifying protein with PALB2 was identified, MRG15 (MORF4L1 gene). Results in human, mouse and C. elegans models delineate molecular and functional relationships with BRCA2, PALB2, RAD51 and RPA1 that suggest a role for MRG15 in the repair of DNA double-strand breaks. Mrg15-deficient murine embryonic fibroblasts showed moderate sensitivity to γ-irradiation relative to controls and reduced formation of Rad51 nuclear foci. Examination of mutants of MRG15 and BRCA2 C. elegans orthologs revealed phenocopy by accumulation of RPA-1 (human RPA1) nuclear foci and aberrant chromosomal compactions in meiotic cells. However, no alterations or mutations were identified for MRG15/MORF4L1 in unclassified FA patients and BrCa familial cases. Finally, no significant associations between common MORF4L1 variants and BrCa risk for BRCA1 or BRCA2 mutation carriers were identified: rs7164529, Ptrend = 0.45 and 0.05, P2df = 0.51 and 0.14, respectively; and rs10519219, Ptrend = 0.92 and 0.72, P2df = 0.76 and 0.07, respectively. CONCLUSIONS: While the present study expands on the role of MRG15 in the control of genomic stability, weak associations cannot be ruled out for potential low-penetrance variants at MORF4L1 and BrCa risk among BRCA2 mutation carriers.
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Neoplasias da Mama/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Neoplasias da Mama/metabolismo , Caenorhabditis elegans , Linhagem Celular , Dano ao DNA , Reparo do DNA , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi , Feminino , Genes BRCA1 , Genes BRCA2 , Predisposição Genética para Doença , Humanos , Camundongos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Fatores de Risco , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Neurofibromatosis Type 1 (NF1) is a genetic condition affecting approximately 1:3500 persons worldwide. The NF1 gene codes for neurofibromin protein, a GTPase activating protein (GAP) and a negative regulator of RAS. The NF1 gene undergoes alternative splicing of exon 23a (E23a) that codes for 21 amino acids placed at the center of the GAP related domain (GRD). E23a-containing type II neurofibromin exhibits a weaker Ras-GAP activity compared to E23a-less type I isoform. Exon E23a has been related with the cognitive impairment present in NF1 individuals. We designed antisense Phosphorodiamidate Morpholino Oligomers (PMOs) to modulate E23a alternative splicing at physiological conditions of gene expression and tested their impact during PC12 cell line neuronal differentiation. Results show that any dynamic modification of the natural ratio between type I and type II isoforms disturbed neuronal differentiation, altering the proper formation of neurites and deregulating both the MAPK/ERK and cAMP/PKA signaling pathways. Our results suggest an opposite regulation of these pathways by neurofibromin and the possible existence of a feedback loop sensing neurofibromin-related signaling. The present work illustrates the utility of PMOs to study alternative splicing that could be applied to other alternatively spliced genes in vitro and in vivo.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Neurofibromatose 1/genética , Neurofibromina 1/genética , Oligonucleotídeos Antissenso/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Éxons/genética , Proteínas Ativadoras de GTPase/genética , Humanos , Neurofibromatose 1/patologia , Neurofibromatose 1/terapia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Oligonucleotídeos Antissenso/genética , Células PC12 , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/genéticaRESUMO
BACKGROUND: Malignant peripheral nerve sheath tumor (MPNST) constitutes the leading cause of neurofibromatosis type 1-related mortality. MPNSTs contain highly rearranged hyperploid genomes and exhibit a high division rate and aggressiveness. We have studied in vitro whether the mitotic kinesins KIF11, KIF15, and KIF23 have a functional role in maintaining MPNST cell survival and can represent potential therapeutic vulnerabilities. METHODS: We studied the expression of kinesin mRNAs and proteins in tumors and cell lines and used several in vitro functional assays to analyze the impact of kinesin genetic suppression (KIF15, KIF23) and drug inhibition (KIF11) in MPNST cells. We also performed in vitro combined treatments targeting KIF11 together with other described MPNST targets. RESULTS: The studied kinesins were overexpressed in MPNST samples. KIF15 and KIF23 were required for the survival of MPNST cell lines, which were also more sensitive than benign control fibroblasts to the KIF11 inhibitors ispinesib and ARRY-520. Co-targeting KIF11 and BRD4 with ARRY-520 and JQ1 reduced MPNST cell viability, synergistically killing a much higher proportion of MPNST cells than control fibroblasts. In addition, genetic suppression of KIF15 conferred an increased sensitivity to KIF11 inhibitors alone or in combination with JQ1. CONCLUSIONS: The mitotic spindle kinesins KIF11 and KIF15 and the cytokinetic kinesin KIF23 play a clear role in maintaining MPNST cell survival and may represent potential therapeutic vulnerabilities. Although further in vivo evidences are still mandatory, we propose a simultaneous suppression of KIF11, KIF15, and BRD4 as a potential therapy for MPNSTs.
RESUMO
BACKGROUND: The aim of this study was to test the feasibility and utility of developing patient-derived orthotopic xenograft (PDOX) models for patients with malignant peripheral nerve sheath tumors (MPNSTs) to aid therapeutic interventions in real time. PATIENT & METHODS: A sporadic relapsed MPNST developed in a 14-year-old boy was engrafted in mice, generating a PDOX model for use in co-clinical trials after informed consent. SNP-array and exome sequencing was performed on the relapsed tumor. Genomics, drug availability, and published literature guided PDOX treatments. RESULTS: A MPNST PDOX model was generated and expanded. Analysis of the patient's relapsed tumor revealed mutations in the MAPK1, EED, and CDK2NA/B genes. First, the PDOX model was treated with the same therapeutic regimen as received by the patient (everolimus and trametinib); after observing partial response, tumors were left to regrow. Regrown tumors were treated based on mutations (palbociclib and JQ1), drug availability, and published literature (nab-paclitaxel; bevacizumab; sorafenib plus doxorubicin; and gemcitabine plus docetaxel). The patient had a lung metastatic relapse and was treated according to PDOX results, first with nab-paclitaxel, second with sorafenib plus doxorubicin after progression, although a complete response was not achieved and multiple metastasectomies were performed. The patient is currently disease free 46 months after first relapse. CONCLUSION: Our results indicate the feasibility of generating MPNST-PDOX and genomic characterization to guide treatment in real time. Although the treatment responses observed in our model did not fully recapitulate the patient's response, this pilot study identify key aspects to improve our co-clinical testing approach in real time.
RESUMO
Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting 1:3,500 individuals. Disease expression is highly variable and complications are diverse. However, currently there is no specific treatment for the disease. NF1 is caused by mutations in the NF1 gene, approximately 2.1% of constitutional mutations identified in our population are deep intronic mutations producing the insertion of a cryptic exon into the mature mRNA. We used antisense morpholino oligomers (AMOs) to restore normal splicing in primary fibroblast and lymphocyte cell lines derived from six NF1 patients bearing three deep intronic mutations in the NF1 gene (c.288+2025T>G, c.5749+332A>G, and c.7908-321C>G). AMOs were designed to target the newly created 5' splice sites to prevent the incorporation of cryptic exons. Our results demonstrate that AMO treatment effectively restored normal NF1 splicing at the mRNA level for the three mutations studied in the different cell lines analyzed. We also found that AMOs had a rapid effect that lasted for several days, acting in a sequence-specific manner and interfering with the splicing mechanism. Finally, to test whether the correction of aberrant NF1 splicing also restored neurofibromin function to wild-type levels, we measured the amount of Ras-GTP after AMO treatment in primary fibroblasts. The results clearly show an AMO-dependent decrease in Ras-GTP levels, which is consistent with the restoration of neurofibromin function. To our knowledge this is the first time that an antisense technique has been used successfully to correct NF1 mutations opening the possibility of a therapeutic strategy for this type of mutation not only for NF1 but for other genetic disorders.