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INTRODUCTION: Circulating tumor DNA (ctDNA) can be obtained from cell-free DNA (cfDNA) andis a new technique for genotyping, response assessment and prognosis in lymphoma. METHODS: Eighteen patients with samples at diagnosis (ctDNA1), after treatment (ctDNA2) and extracted from diagnostic tissue (FFPE) were evaluated. RESULTS: In all patients, at least one mutation in cfDNA was detected at diagnosis. CREBBP was the most frequent mutated gene (67 %). In 12 of the 15 patients with complete remission, the mutation attributed to the disease found at diagnosis cleared with treatment. A reduction in the ctDNA was observed after treatment in 14 patients, 12 of whom achieved complete remission. Correlations were found between the ctDNA at diagnosis and total metabolic tumor volume (r = 0.51; p-value = 0.014) and total lesion glycolysis 2.5 (r = 0.47; p-value = 0.024) by PET at diagnosis and between ctDNA at diagnosis and radiomic features of the lesions with the largest standardized uptake value. There was a strong inverse correlation between ΔctDNA1 and ΔSUVmax by PET/CT (r = -0.8788; p-value = 0.002). CONCLUSION: Analysis of ctDNA and PET/CT in large B-cell lymphoma are complementary data for evaluating tumor burden and tumor clearance after treatment. Analysis of radiomic data might help to identify tumor characteristics and their changes after treatment.
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Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, with few effective treatment strategies. The research on the development of new treatments is often constrained by the limitations of preclinical models, which fail to accurately replicate the disease's essential characteristics. Herein, we describe the obtention, molecular, and functional characterization of the GBM33 cell line. This cell line belongs to the GBM class according to the World Health Organization 2021 Classification of Central Nervous System Tumors, identified by methylation profiling. GBM33 expresses the astrocytic marker GFAP, as well as markers of neuronal origin commonly expressed in GBM cells, such as ßIII-tubulin and neurofilament. Functional assays demonstrated an increased growth rate when compared to the U87 commercial cell line and a similar sensitivity to temozolamide. GBM33 cells retained response to serum starvation, with reduced growth and diminished activation of the Akt signaling pathway. Unlike LN-18 and LN-229 commercial cell lines, GBM33 is able to produce primary cilia upon serum starvation. In summary, the successful establishment and comprehensive characterization of this GBM cell line provide researchers with invaluable tools for studying GBM biology, identifying novel therapeutic targets, and evaluating the efficacy of potential treatments.
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Neoplasias Encefálicas , Glioblastoma , Adulto , Humanos , Glioblastoma/metabolismo , Brasil , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Tubulina (Proteína)/metabolismoRESUMO
Multiple primary thyroid cancer (TC) and breast cancer (BC) are commonly diagnosed, and the lifetime risk for these cancers is increased in patients with a positive family history of both TC and BC. Although this phenotype is partially explained by TP53 or PTEN mutations, a significant number of patients are negative for these alterations. We judiciously recruited patients diagnosed with BC and/or TC having a family history of these tumors and assessed their whole-exome sequencing. After variant prioritization, we selected MUS81 c.1292G>A (p.R431H) for further investigation. This variant was genotyped in a healthy population and sporadic BC/TC tissues and investigated at the protein level and cellular models. MUS81 c.1292G>A was the most frequent variant (25%) and the strongest candidate due to its function of double-strand break repair. This variant was confirmed in four relatives from two families. MUS81 p.R431H protein exhibited lower expression levels in tumors from patients positive for the germline variant, compared with wild-type BC, and normal breast and thyroid tissues. Using cell line models, we showed that c.1292G>A induced protein instability and affected DNA damage response. We suggest that MUS81 is a novel candidate involved in familial BC/TC based on its low frequency in healthy individuals and proven effect in protein stability.
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The p90 ribosomal S6 kinase (RSK) family, a downstream target of Ras/extracellular signal-regulated kinase signaling, can mediate cross-talk with the mammalian target of rapamycin complex 1 pathway. As RSK connects two oncogenic pathways in gliomas, we investigated the protein levels of the RSK isoforms RSK1-4 in nontumoral brain (NB) and grade I-IV gliomas. When compared to NB or low-grade gliomas (LGG), a group of glioblastomas (GBMs) that excluded long-survivor cases expressed higher levels of RSK1 (RSK1hi ). No difference was observed in RSK2 median-expression levels among NB and gliomas; however, high levels of RSK2 in GBM (RSK2hi ) were associated with worse survival. RSK4 expression was not detected in any brain tissues, whereas RSK3 expression was very low, with GBM demonstrating the lowest RSK3 protein levels. RSK1hi and, to a lesser extent, RSK2hi GBMs showed higher levels of phosphorylated RSK, which reveals RSK activation. Transcriptome analysis indicated that most RSK1hi GBMs belonged to the mesenchymal subtype, and RSK1 expression strongly correlated with gene expression signature of immune infiltrates, in particular of activated natural killer cells and M2 macrophages. In an independent cohort, we confirmed that RSK1hi GBMs exclude long survivors, and RSK1 expression was associated with high protein levels of the mesenchymal subtype marker lysosomal protein transmembrane 5, as well as with high expression of CD68, which indicated the presence of infiltrating immune cells. An RSK1 signature was obtained based on differentially expressed mRNAs and validated in public glioma datasets. Enrichment of RSK1 signature followed glioma progression, recapitulating RSK1 protein expression, and was associated with worse survival not only in GBM but also in LGG. In conclusion, both RSK1 and RSK2 associate with glioma malignity, but displaying isoform-specific peculiarities. The progression-dependent expression and association with immune infiltration suggest RSK1 as a potential progression marker and therapeutic target for gliomas.
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Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Proteínas de Membrana/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transcriptoma/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/mortalidade , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Glioblastoma/genética , Glioblastoma/metabolismo , Glioma/genética , Glioma/imunologia , Glioma/secundário , Humanos , Imuno-Histoquímica , Células Matadoras Naturais/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/genética , Gradação de Tumores , Fosforilação , Isoformas de Proteínas , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transcriptoma/genéticaRESUMO
Glioblastoma (GBM) is one of the most aggressive cancers, with median survival of less than 2 years. Despite of considerable advance in molecular classification of GBMs, no improvements in therapy have been described. The scenario is further complicated by tumor heterogeneity and the relationship among genetic, transcriptional and functional findings. Classically, gene expression has been evaluated by steady-state mRNA, however, this does not take translational control into consideration, which contributes considerably to the composition of the proteome. In this study, we evaluated the transcriptomic and translatomic signature of a GBM obtained from a single patient focusing in tumor heterogeneity. In a sampling of eight fragments, we investigated the translation rates, mTORC1 and ERK1/2 pathways and identified both total and polysome associated mRNAs. An increased translation rate was observed in fragments with high-grade histological features. High-grade histology was also associated with the expression of genes related to extracellular matrix (ECM) and angiogenesis, in both transcriptomes and translatomes. However, genes associated with epithelial to mesenchymal transition and stress response, were observed only in translatomes from high-grade fragments. Overall, our results demonstrate that isolation of translated mRNA can be used to identify biomarkers and reveal previously unrecognized determinants of heterogeneity in GBMs.
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Neoplasias do Sistema Nervoso Central/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Linhagem Celular Tumoral , Neoplasias do Sistema Nervoso Central/patologia , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Glioblastoma/patologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Pessoa de Meia-Idade , Biossíntese de Proteínas , RNA Mensageiro/genéticaRESUMO
Background: Human biological material has become an important resource for biomedical research. Tumor Biobanks are facilities that collect, store and distribute samples of tumor and normal tissue for further use in basic and translational cancer research. mRNA-translation has been demonstrated to modulate protein levels and is considered a fundamental post-transcriptional mechanism of gene expression regulation. Thus, determining translation efficiencies of individual mRNAs in human tumors may add another layer of information that contributes to the understanding of tumorigenic pathways. To analyze the RNAs actively engaged in translation, RNAs associated with ribosomes (polysomes) are isolated, identified and compared to total RNA. However, the application of this technique in human tumors depends on the stability of the polysomal structure under Biobank storage conditions that usually consists of ultra-low temperature. Since the effect of freezing on the stability of the polysomal structure in stored tumor samples is not known, it is essential to evaluate this factor in the frozen samples, validating the use of biobank samples in studies of translational efficiency. Methods: Xenograft tumors were divided in two parts, half was subject to immediate processing, and half was frozen for posterior analysis. Both parts were subject to polysomal separation, RNA extraction and identification through RNAseq. Results: It was possible to successfully extract and identify total and polysomal RNA from both fresh and frozen tumoral tissue. The quantification of the polysome profile indicated no difference in the translational efficiency estimated in fresh versus frozen tissue. Gene expression data from the fresh versus frozen tissues were compared and the correlation between the polysome associated fresh x frozen (R = 0,89) and total fresh x frozen (0,90) mRNAs was calculated. No difference was identified between the two conditions. Conclusions: We demonstrated that tissue freezing does not affect the polysomal structure, consequently validating the viability of the use of biobank stored tissue for polysome associated RNA analysis (AU)
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Humanos , Polirribossomos , RNA , Expressão Gênica , Regulação da Expressão Gênica , NeoplasiasRESUMO
O glioblastoma (GBM) está entre os tipos tumorais mais agressivos e de menor resposta a terapias, o que requer, portanto, uma melhor compreensão sobre o comportamento deste tipo tumoral, auxiliando no desenvolvimento de novos tipos de tratamento para a doença. Atualmente, dados de expressão gênica em tumores baseiam-se na população de mRNA total. No entanto, essa abordagem fornece pouca informação sobre os mediadores moleculares das alterações tumorais, pois o nível de expressão de mRNAs não necessariamente reflete os níveis de proteínas expressos. Por outro lado, a população de mRNAs ativamente traduzidos reflete, de maneira mais fidedigna, a expressão proteica, sendo, dessa forma, mais próxima à real medida da expressão gênica. Dito isso, tem-se como objetivo estabelecer a técnica de identificação de RNAs diferencialmente traduzidos (translatômica) e utilizá-la em glioblastomas humanos, contribuindo, assim, para um avanço no conhecimento sobre essa doença. Indica-se com sucesso, como resultado desse estudo, a técnica de translatômica, com o desenvolvimento de um novo gradiente de sacarose que permitiu maior rendimento às preparações e à padronização da identificação de RNAs diferencialmente traduzidos, utilizando tanto microarrays quanto sequenciamento de nova geração. A translatômica foi primeiramente utilizada para comparar a heterogeneidade intratumoral. Comparando áreas histologicamente de maior e menor graus provenientes de um mesmo tumor, observa-se a tradução diferencial de vias de TGF-b e de ciclo celular. Em um segundo momento, a translatômica foi realizada em um número maior de amostras, sendo capaz de classificar três subgrupos moleculares, com um impacto notável na sobrevida: o grupo 1 foi caracterizado pelo aumento das vias associadas à mTORC2, às mitocôndrias à ciliogênese, com sobrevida média de 5 meses; o grupo 2 foi caracterizado por tradução dependente de mTORC1, baixa presença de mitocôndrias e aumento da angiogênese, com uma sobrevida média de 6,3 meses; e o grupo 3 teve uma sobrevida mediana de 21,1 meses e é caracterizado pela alta presença de mitocôndrias, baixa cíliogênese e baixas vias mTORC1 e 2 associadas. Esses grupos não podem ser definidos pela expressão gênica baseada no RNA total, pois não correspondem a classificações moleculares anteriores. Logo, este estudo foi capaz de desenvolver, com sucesso, a técnica de translatômica e aplicá-la ao descobrimento de vias moleculares biologicamente relevantes no processo de tumorigênese em glioblastomas
Glioblastoma (GBM) is among the most aggressive tumor types and the least response to therapy, so better understanding the behavior of this tumor type could help to develop new types of treatment for this disease. Currently, gene expression data on tumors are based on the total mRNA population. However, this approach provides little information on the molecular mediators of tumor changes, since the level of mRNA expression does not necessarily reflect expressed protein levels. Therefore, the population of actively translated mRNAs reflects more accurately the protein expression, being closer to the real measurement of the gene expression. Establish the technique of the identification of differentially translated RNAs (translatomics) and apply it in human glioblastomas, contributing to an advance in the knowledge about this disease. We successfully established the translatomic technique, with the development of a new sucrose gradient that allowed higher yields to the preparations and standardization of the identification of differentially translated RNAs using both microarrays and next generation sequencing. Translatomics was first used to compare intratumoral heterogeneity. Comparing histologically areas of higher and lower degrees from the same tumor, we observed the differential translation of the TGF-b and cell cycle pathways. Afterwards, translatomics were performed in a larger number of samples, being able to classify GBMs into three molecular subgroups, with a remarkable impact on survival. Group 1 was characterized by increased pathways associated with mTORC2, mitochondria and cilia, with an average survival of 5 months. Group 2 was characterized by mTORC1-dependent translation, low mitochondria and increased angiogenesis, with a mean survival of 6.3 months. Group 3 had a median survival of 21.1 months and is characterized by the high presence of mitochondria, low cilia and low associated mTORC1 and 2 pathways. These groups can not be defined using gene expression based on total RNA and thus do not correspond to prior molecular classifications. This study was able to successfully develop the translatomic technique and to apply it to the discovery of biologically relevant molecular pathways in the process of tumorigenesis in glioblastomas
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Humanos , RNA Mensageiro , Expressão Gênica , Inativação Gênica , Modificação Traducional de Proteínas , Glioma/genéticaRESUMO
Prion protein (PrPC) was initially described due to its involvement in transmissible spongiform encephalopathies. It was subsequently demonstrated to be a cell surface molecule involved in many physiological processes, such as vesicle trafficking. Here, we investigated the roles of PrPC in the response to insulin and obesity development. Two independent PrPC knockout (KO) and one PrPC overexpressing (TG20) mouse models were fed high-fat diets, and the development of insulin resistance and obesity was monitored. PrPC KO mice fed high-fat diets presented all of the symptoms associated with the development of insulin resistance: hyperglycemia, hyperinsulinemia, and obesity. Conversely, TG20 animals fed high-fat diets showed reduced weight and insulin resistance. Accordingly, the expression of peroxisome proliferator-activated receptor gamma (PPARγ) was reduced in PrPC KO mice and increased in TG20 animals. PrPC KO cells also presented reduced glucose uptake upon insulin stimulation, due to reduced translocation of the glucose transporter Glut4. Thus, our results suggest that PrPC reflects susceptibility to the development of insulin resistance and metabolic syndrome.
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Transportador de Glucose Tipo 4/metabolismo , Resistência à Insulina , Obesidade/metabolismo , PPAR gama/metabolismo , Proteínas PrPC/metabolismo , Proteínas Priônicas/metabolismo , Células 3T3-L1 , Animais , Membrana Celular/metabolismo , Membrana Celular/patologia , Células Cultivadas , Cruzamentos Genéticos , Dieta Hiperlipídica/efeitos adversos , Embrião de Mamíferos/patologia , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Obesidade/etiologia , Obesidade/patologia , PPAR gama/genética , Proteínas PrPC/genética , Proteínas Priônicas/genética , Transporte Proteico , Aumento de PesoRESUMO
Prion protein (PrP(C)) is a cell surface glycoprotein that is abundantly expressed in nervous system. The elucidation of the PrP(C) interactome network and its significance on neural physiology is crucial to understanding neurodegenerative events associated with prion and Alzheimer's diseases. PrP(C) co-opts stress inducible protein 1/alpha7 nicotinic acetylcholine receptor (STI1/α7nAChR) or laminin/Type I metabotropic glutamate receptors (mGluR1/5) to modulate hippocampal neuronal survival and differentiation. However, potential cross-talk between these protein complexes and their role in peripheral neurons has never been addressed. To explore this issue, we investigated PrP(C)-mediated axonogenesis in peripheral neurons in response to STI1 and laminin-γ1 chain-derived peptide (Ln-γ1). STI1 and Ln-γ1 promoted robust axonogenesis in wild-type neurons, whereas no effect was observed in neurons from PrP(C) -null mice. PrP(C) binding to Ln-γ1 or STI1 led to an increase in intracellular Ca(2+) levels via distinct mechanisms: STI1 promoted extracellular Ca(2+) influx, and Ln-γ1 released calcium from intracellular stores. Both effects depend on phospholipase C activation, which is modulated by mGluR1/5 for Ln-γ1, but depends on, C-type transient receptor potential (TRPC) channels rather than α7nAChR for STI1. Treatment of neurons with suboptimal concentrations of both ligands led to synergistic actions on PrP(C)-mediated calcium response and axonogenesis. This effect was likely mediated by simultaneous binding of the two ligands to PrP(C). These results suggest a role for PrP(C) as an organizer of diverse multiprotein complexes, triggering specific signaling pathways and promoting axonogenesis in the peripheral nervous system.