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MOTIVATION: Over the past decade, network-based approaches have proven useful in identifying disease modules within the human interactome, often providing insights into key mechanisms and guiding the quest for therapeutic targets. This is all the more important, since experimental investigation of potential gene candidates is an expensive task, thus not always a feasible option. On the other hand, many sources of biological information exist beyond the interactome and an important research direction is the design of effective techniques for their integration. RESULTS: In this work, we introduce the Biological Random Walks (BRW) approach for disease gene prioritization in the human interactome. The proposed framework leverages multiple biological sources within an integrated framework. We perform an extensive, comparative study of BRW's performance against well-established baselines. AVAILABILITY AND IMPLEMENTATION: All codes are publicly available and can be downloaded at https://github.com/LeoM93/BiologicalRandomWalks. We used publicly available datasets, details on their retrieval and preprocessing are provided in the Supplementary Material. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Biologia Computacional , Software , HumanosRESUMO
Synonymous mutations continue to be filtered out from most large-scale cancer genome studies, but several lines of evidence suggest they can play driver roles in neoplastic disease. We investigated a case of an aggressive, apparently sporadic medullary thyroid carcinoma (MTC) harboring a somatic RET p.Cys634Arg mutation (a known MTC driver). A germ-line RET substitution (p.Cys630=) had also been found but was considered clinically irrelevant because of its synonymous nature. Next generation sequencing (NGS) of the tumor tissues revealed that the RET mutations were in cis. There was no evidence of gene amplification. Expression analysis found an increase of RET transcript in p.Cys630=;p.Cys634Arg patient compared with that found in 7 MTCs harboring p.Cys634 mutations. Minigene expression assays demonstrated that the presence of the synonymous RET mutation was sufficient to explain the increased RET mRNA level. In silico analyses and RNA immunoprecipitation experiments showed that the p.Cys630 = variant created new exonic splicing enhancer motifs that enhanced SRp55 recruitment to the mutant allele, leading to more efficient maturation of its pre-mRNA and an increased abundance of mature mRNA encoding a constitutively active RET receptor. These findings document a novel mechanism by which synonymous mutations can contribute to cancer progression.
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Carcinoma Neuroendócrino/genética , Proteínas Proto-Oncogênicas c-ret/genética , Neoplasias da Glândula Tireoide/genética , Adulto , Carcinogênese/genética , Elementos Facilitadores Genéticos/genética , Éxons/genética , Mutação em Linhagem Germinativa , Humanos , Masculino , Mutação , Mutação de Sentido Incorreto/genética , Oncogenes , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Splicing de RNA/genética , Análise de Sequência de DNA , Fatores de Processamento de Serina-Arginina/genética , Mutação Silenciosa/genéticaRESUMO
The incidence of papillary thyroid microcarcinoma (microPTC) has dramatically increased in the last decades. Most of these tumors remain small and clinically "silent", only small number progress. Although thyroid surgery used to be the only therapeutic approach, recent guidelines now consider active surveillance for low-risk microPTC. For this reason, more accurate risk stratification of microPTC is needed. The optimal management of low-risk microPTC through accurate risk stratification represents a major clinical issue.
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Carcinoma Papilar/diagnóstico , Carcinoma Papilar/terapia , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/terapia , Carcinoma Papilar/patologia , Carcinoma Papilar/cirurgia , Humanos , Estadiamento de Neoplasias , Fatores de Risco , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/cirurgia , TireoidectomiaRESUMO
Whole exome sequencing (WES) was used to investigate two Italian siblings with wild-type RET genotype, who developed medullary thyroid cancers (MTCs) and, later, primary prostate and breast cancers, respectively. The proband's MTC harbored a p.Met918Thr RET mutation; his sister's MTC was RET/RAS wild-type. Both siblings had a germline mutation (p.Arg417Gln) in the extracellular Sema domain of the proto-oncogene MET. Experiments involving ectopic expression of MET p.Arg417Gln in MET-negative T47D breast cancer cells documented the mutant receptor's functionality and its ability to enhance cell migration and invasion. Our findings highlight a possible link between MET germline mutations and MTCs and suggest that MET p. Arg417Gln may promote an invasive malignant phenotype. The possibility that MTC can be driven/co-driven by a MET mutation has potential management implications, since the tyrosine-kinase inhibitor cabozantinib-approved for treating advanced MTCs-is a specific MET inhibitor.
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Carcinoma Neuroendócrino/genética , Sequenciamento do Exoma , Células Germinativas/metabolismo , Mutação/genética , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-ret/genética , Irmãos , Neoplasias da Glândula Tireoide/genética , Sequência de Bases , Feminino , Humanos , Masculino , Linhagem , Proto-Oncogene MasRESUMO
BACKGROUND: Reporting clinically actionable incidental genetic findings in the course of clinical exome testing is recommended by the American College of Medical Genetics and Genomics (ACMG). However, the performance of clinical exome methods for reporting small subsets of genes has not been previously reported. METHODS: In this study, 57 exome data sets performed as clinical (n = 12) or research (n = 45) tests were retrospectively analyzed. Exome sequencing data was examined for adequacy in the detection of potentially pathogenic variant locations in the 56 genes described in the ACMG incidental findings recommendation. All exons of the 56 genes were examined for adequacy of sequencing coverage. In addition, nucleotide positions annotated in HGMD (Human Gene Mutation Database) were examined. RESULTS: The 56 ACMG genes have 18 336 nucleotide variants annotated in HGMD. None of the 57 exome data sets possessed a HGMD variant. The clinical exome test had inadequate coverage for >50% of HGMD variant locations in 7 genes. Six exons from 6 different genes had consistent failure across all 3 test methods; these exons had high GC content (76%-84%). CONCLUSIONS: The use of clinical exome sequencing for the interpretation and reporting of subsets of genes requires recognition of the substantial possibility of inadequate depth and breadth of sequencing coverage at clinically relevant locations. Inadequate depth of coverage may contribute to false-negative clinical exome results.
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DNA/genética , Exoma/genética , Achados Incidentais , Técnicas de Diagnóstico Molecular/métodos , Análise de Sequência de DNA/métodos , Bases de Dados Genéticas , Variação Genética , Humanos , Estudos RetrospectivosRESUMO
OBJECTIVE: We report the case of a female patient with multiple endocrine neoplasia type 2A (MEN2A) who was found to have a double mutation in the RET (rearranged during transfection) proto-oncogene. METHODS: RET mutational analysis was performed by Sanger DNA sequencing. RESULTS: The proband was a compound heterozygote for the RET germline mutations Val648Ile and Val804Leu on exons 11 and 14, respectively. Genetic analysis of family members showed the presence of the Val648Ile mutation in all except 1 daughter who carried the Val804Leu mutation. However, none of them showed any clinical, biochemical, or histologic signs of neoplastic disease either in the thyroid or adrenal gland. Furthermore, a daughter and the proband's sister who underwent a prophylactic thyroidectomy did not show pathologic evidence of C-cell disease. CONCLUSIONS: We hypothesize that the combined effect of the 2 mutations may have induced the development of pheochromocytoma (PHEO) in our patient. Thus, in the presence of single RET-induced mild medullary thyroid cancer (MTC) phenotype, the search for additional genetic anomalies may lead to the discovery of rare but potentially more aggressive double mutation genotypes.
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Neoplasias das Glândulas Suprarrenais/genética , Substituição de Aminoácidos , Mutação em Linhagem Germinativa , Neoplasia Endócrina Múltipla Tipo 2a/genética , Feocromocitoma/genética , Proteínas Proto-Oncogênicas c-ret/genética , Neoplasias das Glândulas Suprarrenais/patologia , Sequência de Bases , Análise Mutacional de DNA , Feminino , Humanos , Isoleucina/genética , Leucina/genética , Pessoa de Meia-Idade , Dados de Sequência Molecular , Neoplasia Endócrina Múltipla Tipo 2a/patologia , Linhagem , Feocromocitoma/patologia , Proto-Oncogene Mas , Valina/genéticaRESUMO
Thyroid nodules, with a prevalence of almost 25% in the general population, are a common occurrence. Their prevalence varies considerably depending on demographics such as age and sex as well as the presence of risk factors. This article provides a comprehensive overview of the prevalence, risk stratification and current management strategies for thyroid nodules, with a particular focus on changes in diagnostic and therapeutic protocols that have occurred over the past 10 years. Several sonography-based stratification systems (such as Thyroid Imaging Reporting and Data Systems (TIRADS)) might help to predict the malignancy risk of nodules, potentially eliminating the need for biopsy in many instances. However, large or suspicious nodules necessitate cytological evaluation following fine-needle aspiration biopsy for accurate classification. In the case of cytology yielding indeterminate results, additional tools, such as molecular testing, can assist in guiding the management plan. Surgery is no longer the only treatment for symptomatic or malignant nodules: active surveillance or local ablative treatments might be beneficial for appropriately selected patients. To enhance clinician-patient interactions and discussions about diagnostic options, shared decision-making tools have been developed. A personalized, risk-based protocol promotes high-quality care while minimizing costs and unnecessary testing.
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PURPOSE: In this study, we evaluated the biological role of miRNA-31-5p in papillary thyroid cancer (PTC). METHODS: By using the real-time PCR, we measured miRNA-31-5p expression levels in 25 PTC tissues and in two human PTC cell lines (K1 and TPC-1). Then, K1 cells were transiently transfected with mirVana inhibitor or mirVana mimic to miRNA-31-5-p. Cell proliferation was determined by MTT and colony formation assays. The in vitro metastatic ability of thyroid cancer cells was evaluated by adhesion, migration and invasion assays. Epithelial mesenchymal transition (EMT) and Hippo pathway related gene and protein levels were evaluated by using the TaqMan™ Gene Expression Assays and western blot analysis, respectively. RESULTS: We found a significant increase of miR-31-5-p expression in tumor tissue and in K1 cells harboring the BRAF p.V600E mutation. Knockdown of miR-31-5p determined a reduction of cell proliferation, associated with a significant decrease in cell adhesion, migration and invasion properties. A downregulation of EMT markers and YAP/ß-catenin axis was also observed. CONCLUSIONS: Our findings suggest that miRNA-31-5p acts as oncogenic miRNA in human thyrocytes and its overexpression may be involved in the BRAF-related tumorigenesis in PTCs, providing new understanding into its pathological role in PTC progression and invasiveness.
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MicroRNAs , Neoplasias da Glândula Tireoide , Humanos , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias da Glândula Tireoide/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Proliferação de Células/genética , Invasividade Neoplásica/genética , Regulação Neoplásica da Expressão GênicaRESUMO
Aberrant activation of the RET proto-oncogene is implicated in a plethora of cancers. RET gain-of-function point mutations are driver events in multiple endocrine neoplasia 2 (MEN2) syndrome and in sporadic medullary thyroid cancer, while RET rearrangements are driver events in several non-medullary thyroid cancers. Drugs able to inhibit RET have been used to treat RET-mutated cancers. Multikinase inhibitors were initially used, though they showed modest efficacy and significant toxicity. However, new RET selective inhibitors, such as selpercatinib and pralsetinib, have recently been tested and have shown good efficacy and tolerability, even if no direct comparison is yet available between multikinase and selective inhibitors. The advent of high-throughput technology has identified cancers with rare RET alterations beyond point mutations and fusions, including RET deletions, raising questions about whether these alterations have a functional effect and can be targeted by RET inhibitors. In this mini review, we focus on tumors with RET deletions, including deletions/insertions (indels), and their response to RET inhibitors.
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Here, we describe a protocol to generate organoids from human thyroid cancer cells. Starting from the same patient-derived cells, we establish both organoids and primary lines. The organoid medium is supplemented with conditioned medium obtained from the primary cell line. This modification enables culture of the organoid lines for up to 10 months. Even after long-term culture, the organoids retain the genetic and phenotypic characteristics of their tissue of origin.
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Organoides , Neoplasias da Glândula Tireoide , Meios de Cultivo Condicionados/metabolismo , Humanos , Neoplasias da Glândula Tireoide/genéticaRESUMO
PURPOSE: In this study, we investigated the profile of microRNAs (miRNAs) contained in exosomes secreted in the serum of patients with papillary thyroid cancer (PTC). METHODS: Exosome were isolated by adding ExoQuick Exosome Precipitation Solution. Dynamic light scattering (DLS) and western blotting analysis were used to ensure the quality of exosomes. The expression levels of miRNAs were investigated using custom-designed TaqMan Advanced miRNA Array Cards in the screening cohort and using specific TaqMan Advanced MicroRNA Assays in the validation cohort. RESULTS: We identified miR24-3p, miR146a-5p, miR181a-5p and miR382-5p with different expression levels in two different series of 56 and 58 PTC patients as compared with healthy controls. Significant differences in the expression of three PTC exosomal miRNAs, depending on the presence of lymph node metastasis, were detected in only one PTC series. When comparing the expression levels of some PTC-specific exosomal miRNAs with those of the same miRNAs circulating free of any encapsulation, we found a significant correlation for only miR24-3p, suggesting that only select miRNAs are secreted in exosomes. CONCLUSIONS: Our findings demonstrate that four miRNAs are differently secreted in the exosomes of PTC patients, whereas no conclusive results were found to characterize PTCs with lymph node metastasis, suggesting caution in the use of circulating exosomal miRNA expression levels as lymph node metastasis biomarkers. Further investigation into the mechanisms governing miRNA secretion in tumor cells are required.
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MicroRNA Circulante , Exossomos , MicroRNAs , Neoplasias da Glândula Tireoide , Exossomos/metabolismo , Humanos , MicroRNAs/genética , Câncer Papilífero da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologiaRESUMO
The release of molecules in exosomal cargoes is involved in tumor development and progression. We compared the profiles of exosomal microRNAs released by two thyroid cancer cell lines (TPC-1 and K1) with that of non-tumorigenic thyroid cells (Nthy-ori-3-1), and we explored the network of miRNA-target interaction. After extraction and characterization of exosomes, expression levels of microRNAs were investigated using custom TaqMan Advanced array cards, and compared with those expressed in the total cell extracts. The functional enrichment and network-based analysis of the miRNAs' targets was also performed. Five microRNAs (miR-21-5p, miR-31-5p, miR-221-3p, miR-222-3p, and let-7i-3p) were significantly deregulated in the exosomes of tumor cells vs. non-tumorigenic cells, and three of them (miR-31-5p, miR-222-3p, and let-7i-3p) in the more aggressive K1 compared to TPC-1 cells. The network analysis of the five miRNAs identified some genes as targets of more than one miRNAs. These findings permitted the identification of exosomal microRNAs secreted by aggressive PTC cells, and indicated that their main targets are regulators of the tumor microenvironment. A deeper analysis of the functional role of the targets of exosomal miRNAs will provide further information on novel targets of molecular treatments for these neoplasms.
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PURPOSE OF REVIEW: The prevalence of thyroid nodules in the general population is high but only about 5% are malignant lesions. Cytology is usually appropriate to rule out malignancy in sonographically suspicious nodules but in many cases, reports are indeterminate. Molecular testing is a more recent approach to rule out malignancy and guide subsequent management. RECENT FINDINGS: Although several different molecular testing approaches have proven useful in reducing unnecessary surgery, there are still several remaining issues, such as the possible occurrence of RAS mutations (which are difficult to interpret in clinical management) and the role of molecular analysis in specific histotypes, such as Hürthle cell carcinomas. Furthermore, conclusive evidence is lacking regarding the cost-effectiveness and appropriateness of surgical options following molecular tests. SUMMARY: To be useful in clinical practice, molecular tests should be applied to appropriate candidates. In truly uncertain thyroid nodules in which diagnostic surgery may be considered, molecular testing may change the clinical approach and 'save' a number of thyroids.
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Neoplasias da Glândula Tireoide , Nódulo da Glândula Tireoide , Biópsia por Agulha Fina , Humanos , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/genética , Nódulo da Glândula Tireoide/diagnóstico , Nódulo da Glândula Tireoide/genéticaRESUMO
BACKGROUND: Understanding the molecular mechanisms underlying papillary thyroid cancer (PTC) proved to be vital not only for diagnostic purposes but also for tailored treatments. Despite the strong evidence of heritability, only a small subset of alterations has been implicated in PTC pathogenesis. To this reason, we used targeted next-generation sequencing (NGS) to identify candidate variants implicated in PTC pathogenesis, progression, and invasiveness. METHODS: A total of 42 primary PTC tissues were investigated using a targeted next-generation sequencing (NGS) panel enlisting 47 genes involved in DNA repair and tumor progression. RESULTS: We identified 57 point mutations in 78.5% of samples (n = 32). Thirty-two somatic mutations were identified exclusively in known thyroid cancer genes (BRAF, KRAS, NRAS, and TERT). Unpredictably, 45% of the all identified mutations (n = 25) resulted to be germline, most affecting DNA repair genes. Interestingly, none of the latter variants was in the main population databases. Following ACMG classification, 20% of pathogenic/likely pathogenic and 68% of variant of unknown significance were identified. CONCLUSIONS: Overall, our results support the hypothesis that rare germline variants in DNA repair genes are accountable for PTC susceptibility. More data, including the segregation analysis in affected families, should be collected before definitely annotate these alterations and to establish their potential prognostic and treatment implications.
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Neoplasias da Glândula Tireoide , Reparo do DNA/genética , Células Germinativas , Mutação em Linhagem Germinativa , Humanos , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genéticaRESUMO
Cancer stem-like cells (CSCs) have self-renewal abilities responsible for cancer progression, therapy resistance, and metastatic growth. The glioblastoma stem-like cells are the most studied among CSC populations. A recent study identified four transcription factors (SOX2, SALL2, OLIG2, and POU3F2) as the minimal core sufficient to reprogram differentiated glioblastoma (GBM) cells into stem-like cells. Transcriptomic data of GBM tissues and cell lines from two different datasets were then analyzed by the SWItch Miner (SWIM), a network-based software, and FOSL1 was identified as a putative regulator of the previously identified minimal core. Herein, we selected NTERA-2 and HEK293T cells to perform an in vitro study to investigate the role of FOSL1 in the reprogramming mechanisms. We transfected the two cell lines with a constitutive FOSL1 cDNA plasmid. We demonstrated that FOSL1 directly regulates the four transcription factors binding their promoter regions, is involved in the deregulation of several stemness markers, and reduces the cells' ability to generate aggregates increasing the extracellular matrix component FN1. Although further experiments are necessary, our data suggest that FOSL1 reprograms the stemness by regulating the core of the four transcription factors.
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Reprogramação Celular/genética , Células-Tronco Neoplásicas/fisiologia , Proteínas Proto-Oncogênicas c-fos/fisiologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Diferenciação Celular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Células HEK293 , Células HeLa , Humanos , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas c-fos/genéticaRESUMO
The SARS-CoV-2 pandemic is a worldwide public health emergency. Despite the beginning of a vaccination campaign, the search for new drugs to appropriately treat COVID-19 patients remains a priority. Drug repurposing represents a faster and cheaper method than de novo drug discovery. In this study, we examined three different network-based approaches to identify potentially repurposable drugs to treat COVID-19. We analyzed transcriptomic data from whole blood cells of patients with COVID-19 and 21 other related conditions, as compared with those of healthy subjects. In addition to conventionally used drugs (e.g., anticoagulants, antihistaminics, anti-TNFα antibodies, corticosteroids), unconventional candidate compounds, such as SCN5A inhibitors and drugs active in the central nervous system, were identified. Clinical judgment and validation through clinical trials are always mandatory before use of the identified drugs in a clinical setting.
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Tratamento Farmacológico da COVID-19 , COVID-19 , Simulação por Computador , Reposicionamento de Medicamentos , Anti-Inflamatórios/farmacologia , COVID-19/prevenção & controle , Fármacos do Sistema Nervoso Central/farmacologia , Reposicionamento de Medicamentos/métodos , Reposicionamento de Medicamentos/tendências , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica/métodos , Humanos , Fatores Imunológicos/farmacologia , Resultado do Tratamento , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologiaRESUMO
CONTEXT: In multiple endocrine neoplasia (MEN), rearranged during transfection (RET), gene testing has been extensively exploited to characterize tumour aggressiveness and optimize the diagnostic and clinical management. OBJECTIVE: To report the underlying genetic alterations in an unusual case of MEN type 2 (MEN-2A). DESIGN AND PATIENT: Occult medullary thyroid carcinoma (MTC) was diagnosed in a 44-year-old man who had presented with unilateral phaeochromcytoma. DNA extracted from the blood and tumour tissues was analysed for mutations in RET. The transforming potential and mitogenic properties of the identified RET mutation were investigated. RESULTS: The patient carried a novel heterozygous germ-line RET mutation in exon 5 (Val292Met, GTG>ATG) (V292M/RET) with no evidence of additional somatic alterations. The mutation maps to the third cadherin-like domain of RET, which is usually not included in RET screening. Interestingly, MTC with concomitant phaeochromcytoma has never been associated with a RET mutation involving the extracellular cadherin-like domain. V292M/RET was absent in the only two relatives examined. In vitro assays indicate that the mutant has low-grade transforming potential. CONCLUSIONS: Complete characterization and classification of all novel RET mutations are essential for extending genetic analysis in clinical practice. Our findings suggest that: (i) in all MEN-2 patients negative for RET hot-spot mutations, testing should be extended to all coding regions of the gene and (ii) the newly identified V292M/RET mutation is characterized by relatively weak in vitro transforming ability.
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Neoplasias das Glândulas Suprarrenais/genética , Mutação em Linhagem Germinativa , Neoplasia Endócrina Múltipla Tipo 2a/genética , Feocromocitoma/genética , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/fisiologia , Adulto , Animais , Carcinoma Neuroendócrino , Humanos , Masculino , Camundongos , Células NIH 3T3 , Neoplasias da Glândula Tireoide/genéticaRESUMO
KlNDE1 and KlNDI1 code for two inner mitochondrial membrane transdehydrogenases involved in the maintenance of the intracellular NAD(P)H redox balance. The function of these genes during the utilization of fermentative and respiratory carbon sources was studied. During growth in glucose, deletion of KlNDE1 and KlNDI1 led to an altered kinetic of ethanol and glycerol accumulation compared with the wild type; in addition, KlndiDelta was unable to grow in respiratory substrates. Northern analysis and GFP-fusion experiments showed that KlNDE1 and KlNDI1 regulate the expression of KlGUT2, a component of the glycerol-3-phosphate shuttle. Moreover, both genes seem to be involved in the biogenesis of the mitochondrial tubular network.