RESUMO
N6-methyladenosine (m6A) modification of mRNA is emerging as an important regulator of gene expression that affects different developmental and biological processes, and altered m6A homeostasis is linked to cancer1-5. m6A modification is catalysed by METTL3 and enriched in the 3' untranslated region of a large subset of mRNAs at sites close to the stop codon5. METTL3 can promote translation but the mechanism and relevance of this process remain unknown1. Here we show that METTL3 enhances translation only when tethered to reporter mRNA at sites close to the stop codon, supporting a mechanism of mRNA looping for ribosome recycling and translational control. Electron microscopy reveals the topology of individual polyribosomes with single METTL3 foci in close proximity to 5' cap-binding proteins. We identify a direct physical and functional interaction between METTL3 and the eukaryotic translation initiation factor 3 subunit h (eIF3h). METTL3 promotes translation of a large subset of oncogenic mRNAs-including bromodomain-containing protein 4-that is also m6A-modified in human primary lung tumours. The METTL3-eIF3h interaction is required for enhanced translation, formation of densely packed polyribosomes and oncogenic transformation. METTL3 depletion inhibits tumorigenicity and sensitizes lung cancer cells to BRD4 inhibition. These findings uncover a mechanism of translation control that is based on mRNA looping and identify METTL3-eIF3h as a potential therapeutic target for patients with cancer.
Assuntos
Carcinogênese , Fator de Iniciação 3 em Eucariotos/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Metiltransferases/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Animais , Linhagem Celular Tumoral , Ciclização , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Conformação de Ácido Nucleico , Polirribossomos/química , Polirribossomos/metabolismo , Ligação Proteica , RNA Mensageiro/genéticaRESUMO
BACKGROUND: RAS-to-ERK signaling is crucial for the onset and progression of advanced thyroid carcinoma, and blocking ERK dimerization provides a therapeutic benefit in several human carcinomas. Here we analyzed the effects of DEL-22379, a relatively specific ERK dimerization inhibitor, on the activation of the RAS-to-ERK signaling cascade and on tumor-related processes in vitro and in vivo. METHODS: We used a panel of four human anaplastic thyroid carcinoma (ATC) cell lines harboring BRAF or RAS mutations to analyze ERK dynamics and tumor-specific characteristics. We also assessed the impact of DEL-22379 on the transcriptional landscape of ATC cell lines using RNA-sequencing and evaluated its therapeutic efficacy in an orthotopic mouse model of ATC. RESULTS: DEL-22379 impaired upstream ERK activation in BRAF- but not RAS-mutant cells. Cell viability and metastasis-related processes were attenuated by DEL-22379 treatment, but mostly in BRAF-mutant cells, whereas in vivo tumor growth and dissemination were strongly reduced for BRAF-mutant cells and mildly reduced for RAS-mutant cells. Transcriptomics analyses indicated that DEL-22379 modulated the transcriptional landscape of BRAF- and RAS-mutant cells in opposite directions. CONCLUSIONS: Our findings establish that BRAF- and RAS-mutant thyroid cells respond differentially to DEL-22379, which cannot be explained by the previously described mechanism of action of the inhibitor. Nonetheless, DEL-22379 demonstrated significant anti-tumor effects against BRAF-mutant cells in vivo with an apparent lack of toxicity, making it an interesting candidate for the development of combinatorial treatments. Our data underscore the differences elicited by the specific driver mutation for thyroid cancer onset and progression, which should be considered for experimental and clinical approaches.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Animais , Linhagem Celular Tumoral , Dimerização , Humanos , Camundongos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Multimerização Proteica , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Carcinoma Anaplásico da Tireoide/tratamento farmacológico , Carcinoma Anaplásico da Tireoide/genética , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genéticaRESUMO
BACKGROUND: Adenosine deaminases acting on RNA (ADARs) modify many cellular RNAs by catalyzing the conversion of adenosine to inosine (A-to-I), and their deregulation is associated with several cancers. We recently showed that A-to-I editing is elevated in thyroid tumors and that ADAR1 is functionally important for thyroid cancer cell progression. The downstream effectors regulated or edited by ADAR1 and the significance of ADAR1 deregulation in thyroid cancer remain, however, poorly defined. METHODS: We performed whole transcriptome sequencing to determine the consequences of ADAR1 deregulation for global gene expression, RNA splicing and editing. The effects of gene silencing or RNA editing were investigated by analyzing cell viability, proliferation, invasion and subnuclear localization, and by protein and gene expression analysis. RESULTS: We report an oncogenic function for CDK13 in thyroid cancer and identify a new ADAR1-dependent RNA editing event that occurs in the coding region of its transcript. CDK13 was significantly over-edited (c.308A > G) in tumor samples and functional analysis revealed that this editing event promoted cancer cell hallmarks. Finally, we show that CDK13 editing increases the nucleolar abundance of the protein, and that this event might explain, at least partly, the global change in splicing produced by ADAR1 deregulation. CONCLUSIONS: Overall, our data support A-to-I editing as an important pathway in cancer progression and highlight novel mechanisms that might be used therapeutically in thyroid and other cancers.
Assuntos
Adenosina Desaminase/metabolismo , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Regulação Neoplásica da Expressão Gênica , Edição de RNA , Proteínas de Ligação a RNA/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Alelos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Sobrevivência Celular/genética , Progressão da Doença , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Transporte Proteico , Splicing de RNA , Neoplasias da Glândula Tireoide/patologiaRESUMO
Organization of epithelial cells during follicular lumen formation is crucial for thyroid morphogenesis and function of the thyroid gland; however, the molecular mechanisms underlying this are poorly understood. To investigate this process, we established three-dimensional (3D) epithelial culture model systems using Fischer rat thyroid (FRT) cells or murine primary thyrocytes that developed polarized spherical structures with a central lumen, mimicking thyroid follicles. Using microarray-based differential expression analysis of FRT cells grown under 2D or 3D conditions, followed by RNA-mediated interference (RNAi) and morphogenetic analysis, we identified a key role for the thyroid transcription factor Pax8 and its target cadherin-16 (Cdh16) in the generation of polarized follicle-like structures. Silencing Pax8 expression inhibited the acquisition of apical-basal membrane polarity and impaired lumen formation. Both laminin and ß1-integrin (Itgb1) expression was reduced, and cell cytoskeleton polarized distribution was altered. Silencing Cdh16 expression also led to the formation of defective structures characterized by very low laminin expression at the follicle-matrix interface, downregulation of Itgb1, and unpolarized distribution of cell cytoskeleton. Our results demonstrate that Pax8 controls apical-basal follicular polarization and follicle formation through Cdh16.
Assuntos
Polaridade Celular , Células Epiteliais da Tireoide/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno/farmacologia , Regulação para Baixo/efeitos dos fármacos , Combinação de Medicamentos , Integrina beta1/metabolismo , Laminina/metabolismo , Laminina/farmacologia , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fator de Transcrição PAX8 , Proteoglicanas/farmacologia , Ratos , Células Epiteliais da Tireoide/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Thyroid cancer is the most heritable cancer of all those not displaying typical Mendelian inheritance. However, most of the genetic factors that would explain the high heritability remain unknown. Our aim was to identify additional common genetic variants associated with susceptibility to this disease. In order to do so, we performed a genome-wide association study in a series of 398 cases and 502 controls from Spain, followed by a replication in four well-defined Southern European case-control collections contributing a total of 1,422 cases and 1,908 controls. The association between the variation at the 9q22 locus near FOXE1 and thyroid cancer risk was consistent across all series, with several SNPs identified (rs7028661: OR = 1.64, p = 1.0 × 10(-22) , rs7037324: OR = 1.54, p = 1.2 × 10(-17) ). Moreover, the rare alleles of three SNPs (rs2997312, rs10788123 and rs1254167) at 10q26.12 showed suggestive evidence of association with higher risk of the disease (OR = 1.35, p = 1.2 × 10(-04) , OR = 1.26, p = 5.2 × 10(-04) and OR = 1.38, p = 5.9 × 10(-05) , respectively). Finally, the rare allele of rs4075570 at 6q14.1 conferred protection in the series studied (OR = 0.82, p = 2.0 × 10(-04) ). This study suggests that heterogeneity in genetic susceptibility between populations is a key feature to take into account when exploring genetic risk factors related to this disease.
Assuntos
Cromossomos Humanos Par 10/genética , Cromossomos Humanos Par 6/genética , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , Neoplasias da Glândula Tireoide/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Criança , Feminino , Heterogeneidade Genética , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Espanha , Adulto JovemRESUMO
MicroRNA deregulation could be a crucial event in thyroid carcinogenesis. However, current knowledge is based on studies that have used inherently biased methods. Thus, we aimed to define in an unbiased way a list of deregulated microRNAs in well-differentiated thyroid cancer in order to identify diagnostic and prognostic markers. We performed a microRNA deep-sequencing study using the largest well-differentiated thyroid tumor collection reported to date, comprising 127 molecularly characterized tumors with follicular or papillary patterns of growth and available clinical follow-up data, and 17 normal tissue samples. Furthermore, we integrated microRNA and gene expression data for the same tumors to propose targets for the novel molecules identified. Two main microRNA expression profiles were identified: one common for follicular-pattern tumors, and a second for papillary tumors. Follicular tumors showed a notable overexpression of several members of miR-515 family, and downregulation of the novel microRNA miR-1247. Among papillary tumors, top upregulated microRNAs were miR-146b and the miR-221~222 cluster, while miR-1179 was downregulated. BRAF-positive samples displayed extreme downregulation of miR-7 and -204. The identification of the predicted targets for the novel molecules gave insights into the proliferative potential of the transformed follicular cell. Finally, by integrating clinical follow-up information with microRNA expression, we propose a prediction model for disease relapse based on expression of two miRNAs (miR-192 and let-7a) and several other clinicopathological features. This comprehensive study complements the existing knowledge about deregulated microRNAs in the development of well-differentiated thyroid cancer and identifies novel markers associated with recurrence-free survival.
Assuntos
Adenocarcinoma Folicular/genética , Carcinoma/genética , MicroRNAs/genética , Neoplasias da Glândula Tireoide/genética , Adenocarcinoma Folicular/mortalidade , Adolescente , Adulto , Idoso , Carcinoma/mortalidade , Carcinoma Papilar , Análise por Conglomerados , Intervalo Livre de Doença , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/mortalidade , Transcriptoma , Adulto JovemRESUMO
Background: Brain-lung-thyroid syndrome (BLTS) is caused by NKX2-1 haploinsufficiency, resulting in chorea/choreoathetosis, respiratory problems, and hypothyroidism. Genes interacting with NKX2-1 mutants influence its phenotypic variability. We report a novel NKX2-1 missense variant and the modifier function of TAZ/WWTR1 in BLTS. Methods: A child with BLTS underwent next-generation sequencing panel testing for thyroid disorders. His family was genotyped for NKX2-1 variants and screened for germline mosaicism. Mutant NKX2-1 was generated, and transactivation assays were performed on three NKX2-1 target gene promoters. DNA binding capacity and protein-protein interaction were analyzed. Results: The patient had severe BLTS and carried a novel missense variant c.632A>G (p.N211S) in NKX2-1, which failed to bind to specific DNA promoters, reducing their transactivation. TAZ cotransfection did not significantly increase transcription of these genes, although the variant retained its ability to bind to TAZ. Conclusions: We identify a novel pathogenic NKX2-1 variant that causes severe BLTS and is inherited through germline mosaicism. The mutant lacks DNA-binding capacity, impairing transactivation and suggesting that NKX2-1 binding to DNA is essential for TAZ-mediated transcriptional rescue.
Assuntos
Mutação de Sentido Incorreto , Fator Nuclear 1 de Tireoide , Transativadores , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Humanos , Masculino , Fator Nuclear 1 de Tireoide/genética , Fator Nuclear 1 de Tireoide/metabolismo , Transativadores/genética , Ativação Transcricional , Coreia/genética , Fatores de Transcrição/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Atetose , Hipotireoidismo Congênito , Síndrome do Desconforto Respiratório do Recém-NascidoRESUMO
Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant persister cells (DTPs) which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares , Transdução de Sinais , Fatores de Transcrição , Proteínas de Sinalização YAP , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Sinalização YAP/metabolismo , Linhagem Celular Tumoral , Animais , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasia Residual , Camundongos , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Quinase do Linfoma Anaplásico/metabolismo , Quinase do Linfoma Anaplásico/genética , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
IMPORTANCE: BRAF V600E is a prominent oncogene in papillary thyroid cancer (PTC), but its role in PTC-related patient mortality has not been established. OBJECTIVE: To investigate the relationship between BRAF V600E mutation and PTC-related mortality. DESIGN, SETTING, AND PARTICIPANTS: Retrospective study of 1849 patients (1411 women and 438 men) with a median age of 46 years (interquartile range, 34-58 years) and an overall median follow-up time of 33 months (interquartile range, 13-67 months) after initial treatment at 13 centers in 7 countries between 1978 and 2011. MAIN OUTCOMES AND MEASURES: Patient deaths specifically caused by PTC. RESULTS: Overall, mortality was 5.3% (45/845; 95% CI, 3.9%-7.1%) vs 1.1% (11/1004; 95% CI, 0.5%-2.0%) (P < .001) in BRAF V600E-positive vs mutation-negative patients. Deaths per 1000 person-years in the analysis of all PTC were 12.87 (95% CI, 9.61-17.24) vs 2.52 (95% CI, 1.40-4.55) in BRAF V600E-positive vs mutation-negative patients; the hazard ratio (HR) was 2.66 (95% CI, 1.30-5.43) after adjustment for age at diagnosis, sex, and medical center. Deaths per 1000 person-years in the analysis of the conventional variant of PTC were 11.80 (95% CI, 8.39-16.60) vs 2.25 (95% CI, 1.01-5.00) in BRAF V600E-positive vs mutation-negative patients; the adjusted HR was 3.53 (95% CI, 1.25-9.98). When lymph node metastasis, extrathyroidal invasion, and distant metastasis were also included in the model, the association of BRAF V600E with mortality for all PTC was no longer significant (HR, 1.21; 95% CI, 0.53-2.76). A higher BRAF V600E-associated patient mortality was also observed in several clinicopathological subcategories, but statistical significance was lost with adjustment for patient age, sex, and medical center. For example, in patients with lymph node metastasis, the deaths per 1000 person-years were 26.26 (95% CI, 19.18-35.94) vs 5.93 (95% CI, 2.96-11.86) in BRAF V600E-positive vs mutation-negative patients (unadjusted HR, 4.43 [95% CI, 2.06-9.51]; adjusted HR, 1.46 [95% CI, 0.62-3.47]). In patients with distant tumor metastasis, deaths per 1000 person-years were 87.72 (95% CI, 62.68-122.77) vs 32.28 (95% CI, 16.14-64.55) in BRAF V600E-positive vs mutation-negative patients (unadjusted HR, 2.63 [95% CI, 1.21-5.72]; adjusted HR, 0.84 [95% CI, 0.27-2.62]). CONCLUSIONS AND RELEVANCE: In this retrospective multicenter study, the presence of the BRAF V600E mutation was significantly associated with increased cancer-related mortality among patients with PTC. Because overall mortality in PTC is low and the association was not independent of tumor features, how to use BRAF V600E to manage mortality risk in patients with PTC is unclear. These findings support further investigation of the prognostic and therapeutic implications of BRAF V600E status in PTC.
Assuntos
Carcinoma/genética , Carcinoma/mortalidade , Análise Mutacional de DNA , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/mortalidade , Adulto , Idoso , Carcinoma/patologia , Carcinoma Papilar , Feminino , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Mutação , Invasividade Neoplásica , Estudos Retrospectivos , Risco , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/patologiaRESUMO
Thyroid cancer is the most common malignancy of the endocrine system, and its incidence has been steadily increasing. Advances in sequencing have allowed analysis of the entire cancer genome, and has provided new information on the genetic lesions and modifications responsible for the onset, progression, dedifferentiation and metastasis of thyroid carcinomas. Moreover, integrated genomics has advanced our understanding of the development of cancer and its behavior, and has facilitated the identification of new genetic mutations and molecular pathways. The functional analysis of epigenetic modifications, such as DNA methylation, histone acetylation and non-coding RNAs, have contributed to define new regulatory mechanisms that control cell malignancy in thyroid cancer, especially aggressive forms. Here we review the most recent advances in genomics and epigenomics of thyroid cancer, which have resulted in a new classification and interpretation of the initiation and progression of thyroid tumors, providing new tools and opportunities for further investigation and for the clinical development of new treatment strategies.
Assuntos
Epigenômica , Neoplasias da Glândula Tireoide , Humanos , Neoplasias da Glândula Tireoide/genética , Genômica , Metilação de DNA , Epigênese GenéticaRESUMO
BACKGROUND: Loss of the transcription factor GLI-Similar 3 (GLIS3) function causes congenital hypothyroidism (CH) in both humans and mice due to decreased expression of several thyroid hormone (TH) biosynthetic genes in thyroid follicular cells. Whether and to what extent, GLIS3 regulates thyroid gene transcription in coordination with other thyroid transcriptional factors (TFs), such as PAX8, NKX2.1 and FOXE1, is poorly understood. METHODS: PAX8, NKX2.1, and FOXE1 ChIP-Seq analysis with mouse thyroid glands and rat thyrocyte PCCl3 cells was performed and compared to that of GLIS3 to analyze the co-regulation of gene transcription in thyroid follicular cells by these TFs. RESULTS: Analysis of the PAX8, NKX2.1, and FOXE1 cistromes identified extensive overlaps between these TF binding loci and those of GLIS3 indicating that GLIS3 shares many of the same regulatory regions with PAX8, NKX2.1, and FOXE1, particularly in genes associated with TH biosynthesis, induced by thyroid stimulating hormone (TSH), and suppressed in Glis3KO thyroid glands, including Slc5a5 (Nis), Slc26a4, Cdh16, and Adm2. ChIP-QPCR analysis showed that loss of GLIS3 did not significantly affect PAX8 or NKX2.1 binding and did not cause major alterations in H3K4me3 and H3K27me3 epigenetic signals. CONCLUSIONS: Our study indicates that GLIS3 regulates transcription of TH biosynthetic and TSH-inducible genes in thyroid follicular cells in coordination with PAX8, NKX2.1, and FOXE1 by binding within the same regulatory hub. GLIS3 does not cause major changes in chromatin structure at these common regulatory regions. GLIS3 may induce transcriptional activation by enhancing the interaction of these regulatory regions with other enhancers and/or RNA Polymerase II (Pol II) complexes.
RESUMO
BACKGROUND: The transcription factor Pax8 is essential for the differentiation of thyroid cells. However, there are few data on genes transcriptionally regulated by Pax8 other than thyroid-related genes. To better understand the role of Pax8 in the biology of thyroid cells, we obtained transcriptional profiles of Pax8-silenced PCCl3 thyroid cells using whole genome expression arrays and integrated these signals with global cis-regulatory sequencing studies performed by ChIP-Seq analysis RESULTS: Exhaustive analysis of Pax8 immunoprecipitated peaks demonstrated preferential binding to intragenic regions and CpG-enriched islands, which suggests a role of Pax8 in transcriptional regulation of orphan CpG regions. In addition, ChIP-Seq allowed us to identify Pax8 partners, including proteins involved in tertiary DNA structure (CTCF) and chromatin remodeling (Sp1), and these direct transcriptional interactions were confirmed in vivo. Moreover, both factors modulate Pax8-dependent transcriptional activation of the sodium iodide symporter (Nis) gene promoter. We ultimately combined putative and novel Pax8 binding sites with actual target gene expression regulation to define Pax8-dependent genes. Functional classification suggests that Pax8-regulated genes may be directly involved in important processes of thyroid cell function such as cell proliferation and differentiation, apoptosis, cell polarity, motion and adhesion, and a plethora of DNA/protein-related processes. CONCLUSION: Our study provides novel insights into the role of Pax8 in thyroid biology, exerted through transcriptional regulation of important genes involved in critical thyrocyte processes. In addition, we found new transcriptional partners of Pax8, which functionally cooperate with Pax8 in the regulation of thyroid gene transcription. Besides, our data demonstrate preferential location of Pax8 in non-promoter CpG regions. These data point to an orphan CpG island-mediated mechanism that represents a novel role of Pax8 in the transcriptional output of the thyrocyte.
Assuntos
Genoma , Fatores de Transcrição Box Pareados/genética , Glândula Tireoide/metabolismo , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Diferenciação Celular , Linhagem Celular , Imunoprecipitação da Cromatina , Ilhas de CpG , Regulação da Expressão Gênica , Inativação Gênica , Estudo de Associação Genômica Ampla , Células HeLa , Humanos , Análise em Microsséries , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados/antagonistas & inibidores , Fatores de Transcrição Box Pareados/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Simportadores/genética , Simportadores/metabolismo , Glândula Tireoide/citologia , TransfecçãoRESUMO
In order to identify genetic factors related to thyroid cancer susceptibility, we adopted a candidate gene approach. We studied tag- and putative functional SNPs in genes involved in thyroid cell differentiation and proliferation, and in genes found to be differentially expressed in thyroid carcinoma. A total of 768 SNPs in 97 genes were genotyped in a Spanish series of 615 cases and 525 controls, the former comprising the largest collection of patients with this pathology from a single population studied to date. SNPs in an LD block spanning the entire FOXE1 gene showed the strongest evidence of association with papillary thyroid carcinoma susceptibility. This association was validated in a second stage of the study that included an independent Italian series of 482 patients and 532 controls. The strongest association results were observed for rs1867277 (OR[per-allele] = 1.49; 95%CI = 1.30-1.70; P = 5.9x10(-9)). Functional assays of rs1867277 (NM_004473.3:c.-283G>A) within the FOXE1 5' UTR suggested that this variant affects FOXE1 transcription. DNA-binding assays demonstrated that, exclusively, the sequence containing the A allele recruited the USF1/USF2 transcription factors, while both alleles formed a complex in which DREAM/CREB/alphaCREM participated. Transfection studies showed an allele-dependent transcriptional regulation of FOXE1. We propose a FOXE1 regulation model dependent on the rs1867277 genotype, indicating that this SNP is a causal variant in thyroid cancer susceptibility. Our results constitute the first functional explanation for an association identified by a GWAS and thereby elucidate a mechanism of thyroid cancer susceptibility. They also attest to the efficacy of candidate gene approaches in the GWAS era.
Assuntos
Fatores de Transcrição Forkhead/genética , Predisposição Genética para Doença , Variação Genética , Neoplasias da Glândula Tireoide/metabolismo , Fatores Estimuladores Upstream/metabolismo , Adulto , Sequência de Bases , Sítios de Ligação , Estudos de Casos e Controles , Feminino , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Ligação Proteica , Espanha , Neoplasias da Glândula Tireoide/genética , Fatores Estimuladores Upstream/genéticaRESUMO
Background: The Hippo pathway has a fundamental role in tissue homeostasis, but little is known about how this signaling cascade is controlled in the thyroid. PAX8 is an essential driver of thyroid differentiation and is involved in the control of genes crucial for thyroid hormone biosynthesis, including the sodium/iodide symporter (NIS; SLC5A5). A role for the Hippo mediator transcriptional coactivator with PDZ-binding motif (TAZ) as a coactivator of PAX8 to promote thyroglobulin expression has been previously described. Here, we studied the role of TAZ on thyroid differentiation focusing on PAX8-mediated Slc5a5 transcription. Methods: Gene silencing and overexpression assays were performed in rat PCCl3 thyroid follicular cells (TFCs) to determine the role of TAZ in the regulation of Slc5a5. Transcriptional activity of the Hippo mediators was investigated by chromatin immunoprecipitation and promoter-reporter gene activity. Hippo component levels and location were analyzed in PCCl3 cells and in mouse thyroid under different treatment conditions. Results: By suppressing the expression of PAX8 and its binding to the Slc5a5 upstream enhancer, TAZ inhibits Slc5a5 expression, impairing NIS membrane location and activity. Other Hippo effectors such as YAP1 and TEAD1 were not required for the repressor effect of TAZ. We also found an interplay between the Hippo, thyrotropin (TSH), and transforming growth factor ß1 (TGFß) pathways in TFCs. TSH via cyclic adenosine monophosphate activated Hippo signaling pathway and, consequently, TAZ was excluded from the nucleus. We confirmed this in hypothyroid mice, characterized by elevated TSH serum levels, which showed downregulated activation of Hippo signaling in thyroid. Conversely, TAZ nuclear retention was promoted by TGFß, a potent NIS repressor, and TAZ silencing markedly relieved the TGFß-induced inhibition of the symporter. Conclusions: We demonstrate that the effects of TAZ are promoter specific, as it functions as a corepressor of PAX8 to modulate Slc5a5 expression in TFCs. Overall, our data place TAZ as an integrator of the different signaling pathways that control NIS expression, pointing to a role for TAZ in thyroid differentiation and identifying the Hippo pathway as a relevant target to recover NIS levels in thyroid cancer cells.
Assuntos
Iodetos , Simportadores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Iodetos/metabolismo , Camundongos , Fator de Transcrição PAX8/genética , Ratos , Sódio , Simportadores/genética , Simportadores/metabolismo , Tireotropina/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
While the signaling pathways and transcription factors involved in the differentiation of thyroid follicular cells, both in embryonic and adult life, are increasingly well understood, the underlying mechanisms and potential crosstalk between the thyroid transcription factors Nkx2.1, Foxe1 and Pax8 and inductive signals remain unclear. Here, we focused on the transcription factor Sox9, which is expressed in Nkx2.1-positive embryonic thyroid precursor cells and is maintained from embryonic development to adulthood, but its function and control are unknown. We show that two of the main signals regulating thyroid differentiation, TSH and TGFß, modulate Sox9 expression. Specifically, TSH stimulates the cAMP/PKA pathway to transcriptionally upregulate Sox9 mRNA and protein expression, a mechanism that is mediated by the binding of CREB to a CRE site within the Sox9 promoter. Contrastingly, TGFß signals through Smad proteins to inhibit TSH-induced Sox9 transcription. Our data also reveal that Sox9 transcription is regulated by the thyroid transcription factors, particularly Pax8. Interestingly, Sox9 significantly increased the transcriptional activation of Pax8 and Foxe1 promoters and, consequently, their expression, but had no effect on Nkx2.1. Our study establishes the involvement of Sox9 in thyroid follicular cell differentiation and broadens our understanding of transcription factor regulation of thyroid function.
Assuntos
Fatores de Transcrição SOX9/metabolismo , Células Epiteliais da Tireoide/citologia , Células Epiteliais da Tireoide/metabolismo , Glândula Tireoide/metabolismo , Tireotropina/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Camundongos , Fator de Transcrição PAX8/genética , Fator de Transcrição PAX8/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição SOX9/genética , Transdução de Sinais , Glândula Tireoide/citologia , Glândula Tireoide/embriologia , Fator Nuclear 1 de Tireoide/genética , Fator Nuclear 1 de Tireoide/metabolismo , Tireotropina/farmacologia , Transcrição Gênica , Fator de Crescimento Transformador beta/farmacologiaRESUMO
CONTEXT: Circulating microRNAs (miRNAs) are emerging biomarkers of thyroid cancer. OBJECTIVE: This study sought to identify the profile of circulating miRNAs and its response to human recombinant TSH (rhTSH) in thyroid cancer patients with recurrent/persistent disease. METHODS: We obtained serum samples from 30 patients with differentiated thyroid cancer, 14 with recurrent/persistent disease and 16 with complete remission. We used next-generation sequencing to define the miRnomes along with a comprehensive quantitative PCR (qPCR) validation using 2 different platforms. We made a transversal study by comparing serum miRNA profiles of patients with or without recurrent/persistent disease and a longitudinal study looking at differences before and after rhTSH stimulation. Selected miRNAs were then studied in human thyroid cancer cell lines TPC-1, FTC-133, and OCUT-2 in response to TSH stimulation. RESULTS: We could not demonstrate any consistent differences in serum profiles of known miRNAs between patients with and without recurrent/persistent disease or before and after rhTSH stimulation. However, our sequencing data revealed 2 putative novel miRNAs that rise with rhTSH stimulation in the serums of patients with recurrent/persistent disease. We further confirmed by qPCR the upregulation of these putative miRNAs both in serums and in TSH-stimulated cells. We also show miRNAs that are good candidates for housekeeping genes in the serum of patients independently of the levels of TSH. CONCLUSIONS: The present study does not provide evidence that known miRNAs can be used as circulating markers for recurrence of thyroid cancer. However, we suggest that novel miRNA molecules may be related to thyroid cancer pathogenesis.
Assuntos
Adenocarcinoma , MicroRNA Circulante , MicroRNAs , Neoplasias da Glândula Tireoide , Tirotropina Alfa , Biomarcadores , Humanos , Estudos Longitudinais , MicroRNAs/genética , Proteínas Recombinantes , Tireoglobulina , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/genética , Tireotropina/farmacologiaRESUMO
Background: The sodium/iodide symporter (NIS) is a transmembrane protein located on the basolateral membrane of thyrocytes. Despite its physiological and clinical relevance, little is known about the mechanisms that mediate NIS subcellular sorting. In the present study, we examined NIS basolateral trafficking in vitro using non-thyroid and thyroid epithelial cells. Methods: Immunofluorescence and Western blotting were performed to analyze NIS subcellular location and function in cells grown in monolayers under unpolarized and/or polarized conditions. Strategic NIS residues were mutated, and binding of NIS to clathrin adaptor complexes was determined by immunoprecipitation. Results: We show that NIS reaches the plasma membrane (PM) through a thyrotropin-dependent mechanism 24 hours after treatment with the hormone. We demonstrate that NIS basolateral trafficking is a clathrin-mediated mechanism, in which the clathrin adaptor complexes AP-1 (A and B) sort NIS from the trans-Golgi network (TGN) and recycling endosomes (REs). Specifically, we show that the AP-1B µ1 subunit controls NIS basolateral sorting through common REs. In its absence, NIS is apically missorted but remains functional. Additionally, direct NIS basolateral transport from the TGN to the basolateral membrane is mediated by AP-1A through clathrin-coated vesicles that also carry the transferrin receptor. Loss of the µ1 subunit of AP-1A is functionally compensated by AP-1B. Furthermore, loss of both subunits diminishes NIS trafficking to the PM. Finally, we demonstrate that AP-1A binds to the L121 and LL562/563 residues on NIS, whereas AP-1B binds to L583. Conclusions: Our findings highlight the novel involvement of the clathrin-coated machinery in basolateral NIS trafficking. Given that AP-1A expression is reduced in tumors, and its expression correlates with that of NIS, these findings will help uncover new targets in thyroid cancer treatment.
Assuntos
Iodetos , Simportadores , Humanos , Iodetos/metabolismo , Fator de Transcrição AP-1 , Clatrina/metabolismo , Receptores da Transferrina/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Tireotropina/metabolismo , Hormônios , SódioRESUMO
ADARs catalyze adenosine-to-inosine (A-to-I) editing of double-stranded RNA and regulate global gene expression output through interactions with RNA and other proteins. ADARs play important roles in development and disease, and previous work has shown that ADAR1 is oncogenic in a growing list of cancer types. Here we show that ADAR1 is a critical gene for triple-negative breast cancer cells, as ADAR1 loss results in reduced growth (viability and cell cycle progression), invasion, and mammosphere formation. Whole transcriptome sequencing analyses demonstrate that ADAR1 regulates both coding and noncoding targets by altering gene expression level, A-to-I editing, and splicing. We determine that a recoding edit in filamin B (FLNB chr3:58156064) reduces the tumor suppressive activities of the protein to promote growth and invasion. We also show that several tumor suppressor miRNAs are upregulated upon ADAR1 loss and suppress cell-cycle progression and invasion. This work describes several novel mechanisms of ADAR1-mediated oncogenesis in triple-negative breast cancer, providing support to strategies targeting ADAR1 in this aggressive cancer type that has few treatment options. IMPLICATIONS: Targeting ADAR1 and thus downstream FLNB editing and miRNA regulation represents a possible novel therapeutic strategy in triple-negative breast cancer.
Assuntos
MicroRNAs , Neoplasias de Mama Triplo Negativas , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Carcinogênese , Perfilação da Expressão Gênica , Humanos , MicroRNAs/genética , Edição de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
Thyroid cancer is the most common primary endocrine malignancy in adults and its incidence is rapidly increasing. Long non-coding RNAs (lncRNAs), generally defined as RNA molecules longer than 200 nucleotides with no protein-encoding capacity, are highly tissue-specific molecules that serve important roles in gene regulation through a variety of different mechanisms, including acting as competing endogenous RNAs (ceRNAs) that 'sponge' microRNAs (miRNAs). In the present study, using an integrated approach through RNA-sequencing of paired thyroid tumor and non-tumor samples, we have identified an interactome network between lncRNAs and miRNAs and examined the functional consequences in vitro and in vivo of one of such interactions. We have identified a likely operative post-transcriptional regulatory network in which the downregulated lncRNA, SPTY2D1-AS1, is predicted to target the most abundant and upregulated miRNAs in thyroid cancer, particularly miR-221, a well-known oncomiRNA in cancer. Indeed, SPTY2D1-AS1 functions as a potent tumor suppressor in vitro and in vivo, it is downregulated in the most advanced stages of human thyroid cancer, and it seems to block the processing of the primary form of miR-221. Overall, our results link SPTY2D1-AS1 to thyroid cancer progression and highlight the potential use of this lncRNA as a therapeutic target of thyroid cancer.
Assuntos
MicroRNAs , RNA Longo não Codificante , Neoplasias da Glândula Tireoide , Adulto , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , RNA Longo não Codificante/genética , Neoplasias da Glândula Tireoide/genéticaRESUMO
While much is known about Groucho corepressors in Drosophila development, less is known about Grg homologs in mammalian embryogenesis. The transcription factors FoxA1 and FoxA2 are redundantly necessary for liver-inductive competence of the endoderm, and recently we found that FoxA factors bind Grg3, recruit the corepressor to FoxA target genes, and cause transcriptional repression, when Grg3 is ectopically expressed in adult liver cell lines that express little or no endogenous Grg. Unexpectedly, we now find that Grg1 and Grg3 mRNAs are co-expressed with FoxA factors in the foregut endoderm, prior to liver differentiation, though only Grg3 protein is expressed there. Grg3 mRNA and protein are extinguished at the onset of liver differentiation. Lentiviral delivery of Grg3 to explants of foregut endoderm suppresses liver gene induction. We suggest that Grg expression in the endoderm helps suppress the liver program and find that endodermal competence involves a balance between activators and corepressors.