RESUMO
Immune cells constantly survey the host for pathogens or tumors and secrete cytokines to alert surrounding cells of these threats. In vivo, activated immune cells secrete cytokines for several hours, yet an acute immune reaction occurs over days. Given these divergent timescales, we addressed how cytokine-responsive cells translate brief cytokine exposure into phenotypic changes that persist over long timescales. We studied melanoma cell responses to transient exposure to the cytokine interferon γ (IFNγ) by combining a systems-scale analysis of gene expression dynamics with computational modeling and experiments. We discovered that IFNγ is captured by phosphatidylserine (PS) on the surface of viable cells both in vitro and in vivo then slowly released to drive long-term transcription of cytokine-response genes. This mechanism introduces an additional function for PS in dynamically regulating inflammation across diverse cancer and primary cell types and has potential to usher in new immunotherapies targeting PS and inflammatory pathways.
Assuntos
Comunicação Celular , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Interferon gama/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/metabolismo , Fosfatidilserinas/metabolismo , Linfócitos T/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura , Biologia Computacional , Simulação por Computador , Bases de Dados Genéticas , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interferon gama/imunologia , Interleucina-12/imunologia , Interleucina-12/metabolismo , Interleucina-23/imunologia , Interleucina-23/metabolismo , Janus Quinases/metabolismo , Ativação Linfocitária , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilserinas/imunologia , Fosforilação , Células RAW 264.7 , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/imunologia , Neoplasias da Glândula Tireoide/patologia , Fatores de Tempo , Transcrição Gênica , Receptor de Interferon gamaRESUMO
Hormone-dependent activation of enhancers includes histone hyperacetylation and mediator recruitment. Histone hyperacetylation is mostly explained by a bimodal switch model, where histone deacetylases (HDACs) disassociate from chromatin, and histone acetyl transferases (HATs) are recruited. This model builds on decades of research on steroid receptor regulation of transcription. Yet, the general concept of the bimodal switch model has not been rigorously tested genome wide. We have used a genomics approach to study enhancer hyperacetylation by the thyroid hormone receptor (TR), described to operate as a bimodal switch. H3 acetylation, HAT and HDAC ChIP-seq analyses of livers from hypo- and hyperthyroid wildtype, TR deficient and NCOR1 disrupted mice reveal three types of thyroid hormone (T3)-regulated enhancers. One subset of enhancers is bound by HDAC3-NCOR1 in the absence of hormone and constitutively occupy TR and HATs irrespective of T3 levels, suggesting a poised enhancer state in absence of hormone. In presence of T3, HDAC3-NCOR1 dissociates from these enhancers leading to histone hyperacetylation, suggesting a histone acetylation rheostat function of HDACs at poised enhancers. Another subset of enhancers, not occupied by HDACs, is hyperacetylated in a T3-dependent manner, where TR is recruited to chromatin together with HATs. Lastly, a subset of enhancers, is not occupied directly by TR yet requires TR for histone hyperacetylation. This indirect enhancer activation involves co-association with TR bound enhancers within super-enhancers or topological associated domains. Collectively, this demonstrates various mechanisms controlling hormone-dependent transcription and adds significant details to the otherwise simple bimodal switch model.
Assuntos
Elementos Facilitadores Genéticos/efeitos dos fármacos , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Receptores dos Hormônios Tireóideos/genética , Hormônios Tireóideos/farmacologia , Acetilação , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Histona Desacetilases/metabolismo , Fígado/química , Masculino , Camundongos , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismoRESUMO
Patients with mutations of the THRA gene exhibit classical features of hypothyroidism, including erythroid disorders. We previously created a mutant mouse expressing a mutated TRα1 (denoted as PV; Thra1PV/+ mouse) that faithfully reproduces the classical hypothyroidism seen in patients. Using Thra1PV/+ mice, we explored how the TRα1PV mutant acted to cause abnormalities in erythropoiesis. Thra1PV/+ mice exhibited abnormal red blood cell indices similarly as reported for patients. The total bone marrow cells and erythrocytic progenitors were markedly reduced in the bone marrow of Thra1PV/+ mice. In vitro terminal differentiation assays showed a significant reduction of mature erythrocytes in Thra1PV/+ mice. In wild-type mice, the clonogenic potential of progenitors in the erythrocytic lineage was stimulated by thyroid hormone (T3), suggesting that T3 could directly accelerate the differentiation of progenitors to mature erythrocytes. Analysis of gene expression profiles showed that the key regulator of erythropoiesis, the Gata-1 gene, and its regulated genes, such as the Klf1, ß-globin, dematin genes, CAII, band3 and eALAS genes, involved in the maturation of erythrocytes, was decreased in the bone marrow cells of Thra1PV/+ mice. We further elucidated that the Gata-1 gene was a T3-directly regulated gene and that TRα1PV could impair erythropoiesis via repression of the Gata-1 gene and its regulated genes. These results provide new insights into how TRα1 mutants acted to cause erythroid abnormalities in patients with mutations of the THRA gene. Importantly, the Thra1PV/+ mouse could serve as a preclinical mouse model to identify novel molecular targets for treatment of erythroid disorders.
Assuntos
Eritropoese/genética , Fator de Transcrição GATA1/genética , Hipotireoidismo/genética , Receptores alfa dos Hormônios Tireóideos/genética , Animais , Diferenciação Celular/genética , Eritrócitos , Humanos , Hipotireoidismo/fisiopatologia , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Transgênicos , Mutação , Transcriptoma , Tri-Iodotironina/genética , Globinas beta/genéticaRESUMO
The functional unit of the thyroid gland, the thyroid follicle, dynamically responds to various stimuli to maintain thyroid hormone homeostasis. However, thyroid follicles in the adult human thyroid gland have a very limited regenerative capacity following partial resection of the thyroid gland. To gain insight into follicle regeneration in the adult thyroid gland, we observed the regeneration processes of murine thyroid follicles after partial resection of the lower third of the thyroid gland in 10-week-old male C57BL/6 mice. Based on sequential observation of the partially resected thyroid lobe, we found primitive follicles forming in the area corresponding to the central zone of the intact lateral thyroid lobe. The primitive thyroid follicles were multiciliated and had coarsely vacuolated cytoplasm and large vesicular nuclei. Consistently, these primitive follicular cells did not express the differentiation markers paired box gene-8 and thyroid transcription factor-1 (clone SPT24), but were positive for forkhead box protein A2 and leucine-rich repeat-containing G-protein-coupled receptor 4/GPR48. Follicles newly generated from the primitive follicles had clear or vacuolar cytoplasm with dense, darkly stained nuclei. At day 21 after partial thyroidectomy, the tall cuboidal follicular epithelial cells had clear or vacuolar cytoplasm, and the intraluminal colloid displayed pale staining. Smaller activated follicles were found in the central zone of the lateral lobe, whereas larger mature follicles were located in the peripheral zone. Based on these observations, we propose that the follicle regeneration process in the partially resected adult murine thyroid gland associated with the appearance of primitive follicular cells may be a platform for the budding of differentiated follicles in mice.
Assuntos
Regeneração , Glândula Tireoide/citologia , Glândula Tireoide/fisiologia , Tireoidectomia , Adulto , Animais , Cílios/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Receptores Acoplados a Proteínas G/metabolismo , Glândula Tireoide/cirurgia , Hormônios Tireóideos/sangue , Fatores de TempoRESUMO
We have recently identified that phosphorylation at tyrosine (Y)406 is critical for the tumor suppressor functions of the thyroid hormone receptor ß1 (TRß) in a breast cancer line. However, still unclear is whether the critical tumor suppressor role of phosphorylated Y406 of TRß is limited to only breast cancer cells or could be extended to other cell types. In the present studies, we addressed this question by stably expressing TRß, a mutated TRß oncogene (PV), or a TRß mutated at Y406 (TRßY406F) in rat PCCL3 thyroid follicular cells and evaluated their tumor characteristics in athymic mice with elevated thyroid stimulating hormone. PCCL3 cells stably expressing PV (PCCL3-PV), TRßY406F (PCCL3-TRßY406F), or vector only (PCCL3-Neo) developed tumors with sizes in the rank order of TRßY406F>PV = Neo, whereas PCCL3 cells expressing TRß (PCCL3-TRß) barely developed tumors. As evidenced by markedly elevated Ki67, cyclin D1, and p-Rb protein abundance, proliferative activity was high in PV and TRßY406F tumors, but low in TRß tumors. These results indicate that TRß acted as a tumor suppressor in PCCL3 cells, whereas TRßY406F and PV had lost tumor suppressor activity. Interestingly, TRßY406F tumors had very low necrotic areas with decreased TNFα-NFκB signaling to lower apoptotic activity. In contrast, PV tumors had prominent large necrotic areas, with no apparent changes in TNFα-NFκB signaling, indicating distinct oncogenic activities of mutant PV and TRßY406F. Thus, the present studies uncovered a novel mechanism by which TRß could function as a tumor suppressor through modulation of the TNFα-NFκB signaling. © 2016 Wiley Periodicals, Inc.
Assuntos
Mutação Puntual , Glândula Tireoide/patologia , Receptores beta dos Hormônios Tireóideos/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Genes Supressores de Tumor , Humanos , Camundongos , Camundongos Nus , Fosforilação , Ratos , Glândula Tireoide/metabolismo , Receptores beta dos Hormônios Tireóideos/química , Tirosina/análise , Tirosina/genéticaRESUMO
Mutations of the thyroid hormone receptor α gene (THRA) cause hypothyroidism in patients with growth and developmental retardation, and skeletal dysplasia. Genetic evidence indicates that the dominant negative activity of TRα1 mutants underlies pathological manifestations. Using a mouse model of hypothyroidism caused by a dominant negative TRα1PV mutant and its derived mouse model harboring a mutated nuclear receptor corepressor (NCOR1ΔID) (Thra1(PV/+)Ncor1(ΔID/ΔID) mice), we recently showed that aberrant release of TRα1 mutants from the NCOR1 repressor complex mediates dominant negative actions of TRα1 mutants in vivo. We tested the hypothesis that deacetylation of nucleosomal histones associated with aberrant recruitment of corepressors by TRα1 mutants underlies pathological phenotypic expression. We treated Thra1(PV/+)and Thra1(PV/+)Ncor1(ΔID/ΔID) mice with a histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxyamic acid (SAHA). SAHA significantly ameliorated the impaired growth, bone development and adipogenesis of Thra1(PV/+) mice. In Thra1(PV/+)Ncor1(ΔID/ΔID) mice, SAHA improved these abnormalities even further. We focused our molecular analyses on how SAHA improved the impaired adipogenesis leading to the lean phenotype. We found that SAHA reverted the impaired adipogenesis by de-repressing the expression of the two master regulators of adipogenesis, C/ebpα and Pparγ, as well as other adipogenic genes at both the mRNA and protein levels. Chromatin immunoprecipitation analyses indicated SAHA increased the extent of acetylation of nucleosomal H4K5 and H3 to re-activate adipogenic genes to reverting adipogenesis. Thus, HDAC confers in vivo aberrant actions of TRα1 mutants. Importantly, for the first time, the present studies show that HDAC inhibitors are clearly beneficial for hypothyroidism and could be therapeutics for treatment.
Assuntos
Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Hipotireoidismo/tratamento farmacológico , Receptores alfa dos Hormônios Tireóideos/genética , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Avaliação Pré-Clínica de Medicamentos , Epigênese Genética/efeitos dos fármacos , Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Hipotireoidismo/sangue , Hipotireoidismo/genética , Gordura Intra-Abdominal/efeitos dos fármacos , Gordura Intra-Abdominal/patologia , Masculino , Camundongos Transgênicos , PPAR gama/genética , PPAR gama/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Hormônios Tireóideos/sangue , Vorinostat , Aumento de Peso/efeitos dos fármacosRESUMO
Genetic evidence from patients with mutations of the thyroid hormone receptor α gene (THRA) indicates that the dominant negative activity of mutants underlies the pathological manifestations. However, the molecular mechanisms by which TRα1 mutants exert dominant negative activity in vivo are not clear. We tested the hypothesis that the severe hypothyroidism in patients with THRA mutations is due to an inability of TRα1 mutants to properly release the nuclear corepressors (NCORs), thereby inhibiting thyroid hormone-mediated transcription activity. We crossed Thra1(PV) mice, expressing a dominant negative TRα1 mutant (TRα1PV), with mice expressing a mutant Ncor1 allele (Ncor1(ΔID) mice) that cannot recruit the TR or PV mutant. TRα1PV shares the same C-terminal mutated sequences as those of patients with frameshift mutations of the THRA gene. Remarkably, NCOR1ΔID ameliorated abnormalities in the thyroid-pituitary axis of Thra1(PV/+) mice. The severe retarded growth, infertility, and delayed bone development were partially reverted in Thra1(PV/+) mice expressing NCOR1ΔID. The impaired adipogenesis was partially corrected by de-repression of peroxisome-proliferator activated receptor γ and CCAAT/enhancer-binding protein α gene, due to the inability of TRα1PV to recruit NCOR1ΔID to form a repressor complex. Thus, the aberrant recruitment of NCOR1 by TRα1 mutants could lead to clinical hypothyroidism in humans. Therefore, therapies aimed at the TRα1-NCOR1 interaction or its downstream actions could be tested as potential targets in treating TRα1 mutant-mediated hypothyroidism in patients.
Assuntos
Mutação , Correpressor 1 de Receptor Nuclear/fisiologia , Receptores alfa dos Hormônios Tireóideos/metabolismo , Alelos , Animais , Desenvolvimento Ósseo , Cruzamentos Genéticos , Feminino , Mutação da Fase de Leitura , Hipotireoidismo/metabolismo , Hipotireoidismo/fisiopatologia , Infertilidade/patologia , Metabolismo dos Lipídeos , Masculino , Camundongos , Hipófise/metabolismo , Hipófise/patologia , Domínios e Motivos de Interação entre Proteínas , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Tiroxina/metabolismo , Tri-Iodotironina/metabolismoRESUMO
Cancer gender disparity has been observed for a variety of human malignancies. Thyroid cancer is one such cancer with a higher incidence in women, but more aggressive disease in men. There is scant evidence on the role of sex hormones on cancer initiation/progression. Using a transgenic mouse model of follicular thyroid cancer (FTC), we found castration led to lower rates of cancer in females and less advanced cancer in males. Mechanistically, less advanced cancer in castrated males was due to increased expression of tumor suppressor (Glipr1, Sfrp1) and immune-regulatory genes and higher tumor infiltration with M1 macrophages and CD8 cells. Functional study showed that GLIPR1 reduced cell growth and increased chemokine secretion (Ccl5) that activates immune cells. Our data demonstrate that testosterone regulates thyroid cancer progression by reducing tumor suppressor gene expression and tumor immunity.
Assuntos
Adenocarcinoma Folicular/patologia , Genes Supressores de Tumor , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Testosterona/metabolismo , Neoplasias da Glândula Tireoide/patologia , Adenocarcinoma Folicular/genética , Adenocarcinoma Folicular/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Castração , Linhagem Celular , Proliferação de Células , Quimiocina CCL5/metabolismo , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Macrófagos/imunologia , Masculino , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Distribuição por Sexo , Receptores beta dos Hormônios Tireóideos/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/imunologiaRESUMO
BACKGROUND: Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that mediate the actions of the thyroid hormone (T3) in development, growth, and differentiation. The THRA and THRB genes encode several TR isoforms that express in a tissue- and development-dependent manner. In the past decades, a significant advance has been made in the understanding of TR actions in maintaining normal cellular functions. However, the roles of TRs in human cancer are less well understood. The reduced expression of TRs because of hypermethylation, or deletion of TR genes found in human cancers suggests that TRs could function as tumor suppressors. A close association of somatic mutations of TRs with human cancers further supports the notion that the loss of normal functions of TR could lead to uncontrolled growth and loss of cell differentiation. SCOPE OF REVIEW: In line with the findings from association studies in human cancers, mice deficient in total functional TRs (Thra1(-/-)Thrb(-/-) mice) or with a targeted homozygous mutation of the Thrb gene (denoted PV; Thrb(PV/PV) mice) spontaneously develop metastatic thyroid carcinoma. This review will examine the evidence learned from these genetically engineered mice that provided strong evidence to support the critical role of TRs in human cancer. MAJOR CONCLUSIONS: Loss of normal functions of TR by deletion or by mutations could contribute to cancer development, progression and metastasis. GENERAL SIGNIFICANCE: Novel mechanistic insights are revealed in how aberrant TR activities lead to carcinogenesis. Mouse models of thyroid cancer provide opportunities to identify molecular targets as potential treatment modalities. This article is part of a Special Issue entitled Thyroid hormone signalling.
Assuntos
Neoplasias/genética , Neoplasias/metabolismo , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Transdução de Sinais , Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismoRESUMO
Mutations in the ligand-binding domain of the thyroid hormone receptor ß (TRß) lead to resistance to thyroid hormone (RTH). These TRß mutants function in a dominant-negative fashion to interfere with the transcription activity of wild-type thyroid hormone receptors (TRs), leading to dysregulation of the pituitary-thyroid axis and resistance in peripheral tissues. The molecular mechanism by which TRß mutants cause RTH has been postulated to be an inability of the mutants to properly release the nuclear corepressors (NCORs), thereby inhibiting thyroid hormone (TH)-mediated transcription activity. To test this hypothesis in vivo, we crossed Thrb(PV) mice (a model of RTH) expressing a human TRß mutant (PV) with mice expressing a mutant Ncor1 allele (Ncor1(ΔID) mice) that cannot recruit a TR or a PV mutant. Remarkably, in the presence of NCOR1ΔID, the abnormally elevated thyroid-stimulating hormone and TH levels found in Thrb(PV) mice were modestly but significantly corrected. Furthermore, thyroid hyperplasia, weight loss, and other hallmarks of RTH were also partially reverted in mice expressing NCOR1ΔID. Taken together, these data suggest that the aberrant recruitment of NCOR1 by RTH TRß mutants leads to clinical RTH in humans. The present study suggests that therapies aimed at the TR-NCOR1 interaction or its downstream actions could be tested as potential targets in treating RTH.
Assuntos
Correpressor 1 de Receptor Nuclear/fisiologia , Síndrome da Resistência aos Hormônios Tireóideos/genética , Síndrome da Resistência aos Hormônios Tireóideos/fisiopatologia , Animais , Modelos Animais de Doenças , Genes erbA , Humanos , Masculino , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Mutação , Correpressor 1 de Receptor Nuclear/química , Correpressor 1 de Receptor Nuclear/genética , Estrutura Terciária de Proteína , Deleção de Sequência , Receptores beta dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/fisiologia , Síndrome da Resistência aos Hormônios Tireóideos/patologia , Hormônios Tireóideos/sangue , Hormônios Tireóideos/fisiologiaRESUMO
Thyroid hormones (THs) T4 and T3 are vital for development, growth, and metabolism. Thyroid dysfunction can also cause problems in fertility, suggesting involvement of THs in reproduction. In zebrafish, there exist 2 forms of TH receptor alpha gene (thraa and thrab). Disruption of these genes by CRISPR/Cas9 showed no reproductive irregularities in the thraa mutant; however, inactivation of the thrab gene resulted in female infertility. Although young female mutants (thrabm/m) showed normal ovarian development and folliculogenesis before sexual maturation, they failed to release eggs during oviposition after sexual maturation. This spawning failure was due to oviductal blockage at the genital papilla. The obstruction of the oviduct subsequently caused an accumulation of the eggs in the ovary, resulting in severe ovarian hypertrophy, abdominal distention, and disruption of folliculogenesis. Gene expression analysis showed expression of both TH receptors and estrogen receptors in the genital papilla, suggesting a direct TH action and potential interactions between thyroid and estrogen signaling pathways in controlling genital papilla development and function. In addition to their actions in the reproductive tracts, THs may also have direct effects in the ovary, as suggested by follicle atresia and cessation of folliculogenesis in the heterozygous mutant (thrab+/m), which was normal in all aspects of female reproduction in young and sexually mature fish but exhibited premature ovarian failure in aged females. In summary, this study provides substantial evidence for roles of THs in controlling the development and functions of both reproductive tract and ovary.
Assuntos
Infertilidade Feminina , Ovário , Peixe-Zebra , Animais , Feminino , Peixe-Zebra/genética , Infertilidade Feminina/genética , Ovário/metabolismo , Receptores alfa dos Hormônios Tireóideos/genética , Receptores alfa dos Hormônios Tireóideos/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Mutação , Sistemas CRISPR-Cas , Reprodução/genéticaRESUMO
Background: Anaplastic thyroid cancer (ATC) is highly aggressive and has very limited treatment options. Recent studies suggest that cancer stem cell (CSC) activity in ATC could underlie this recurrence and resistance to treatment. The recent approval by the U.S. Food and Drug Administration of the combined treatment of BRAF and MEK inhibitors for ATC patients has shown some efficacy in patients harboring the BRAFV600E mutation. However, it was unknown whether the combined treatment could affect the CSC activity. This study explores the effects of the BRAF and MEK inhibitors on CSC activity in human ATC cells. Methods: Using three human ATC cells, THJ-11T, THJ-16T, and 8505C cells, we evaluated the effects of dabrafenib (a BRAF kinase inhibitor), trametinib (an MEK inhibitor), or a combined treatment of the two drugs on the CSC activity by tumorsphere formation, Aldefluor assays, expression profiles of key CSC markers, immunohistochemistry, and in vivo xenograft mouse models. Furthermore, we also used confocal imaging to directly visualize the effects on drugs on CSCs by the SORE6-mCherry reporter in cultured cells and xenograft tumor cells. Results: The BRAF inhibitor, dabrafenib, had weak efficacy, while the MEK inhibitor, trametinib, showed strong efficacy in attenuating the CSC activity, as evidenced by suppression of CSC marker expression, tumorsphere formation, and Aldefluor assays. Using ATC cells expressing a fluorescent CSC SORE6 reporter, we showed reduction of CSC activity in the rank order of combined > trametinib > dabrafenib through in vitro and in vivo xenograft models. Molecular analyses showed that suppression of CSC activity by these drugs was, in part, mediated by attenuation of the transcription by dampening the RNA polymerase II activity. Conclusions: Our analyses demonstrated the presence of CSCs in ATC cells. The inhibition of CSC activity by the MEK signaling could partially account for the efficacy of the combined treatment shown in ATC patients. However, our studies also showed that not all CSC activity was totally abolished, which may account for the recurrence observed in ATC patients. Our findings have provided new insights into the molecular basis of efficacy and limitations of these drugs in ATC patients.
Assuntos
Imidazóis , Oximas , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Humanos , Camundongos , Animais , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Proteínas Proto-Oncogênicas B-raf/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/uso terapêutico , Células-Tronco Neoplásicas/patologia , Linhagem Celular Tumoral , MutaçãoRESUMO
Anaplastic thyroid cancer (ATC) is a clinically aggressive form of undifferentiated thyroid cancer with limited treatment options. Tumor-associated macrophages (TAMs) constitute over 50% of ATC-infiltrating cells, and their presence is associated with a poor prognosis. We have previously shown that paracrine signals released by ATC cells induced pro-tumor M2-like polarization of human monocytes. However, which soluble factors derived from ATC cells drive monocyte activation, are largely unknown. In this study we investigated the participation of transforming growth factor ß1 (TGFß1) on the phenotype of macrophage activation induced by ATC cell-derived conditioned media (CM). THP-1 cells exposed to CM derived from ATC cells and recombinant human TGFß1 induced M2-like macrophage polarization, showing high CD163 and Dectin1 expression. Moreover, we showed that TGFß1 induced the messenger RNA (mRNA) and protein expression of the transcription factors SNAIL and SLUG. Accordingly, increased TGFß1 secretion from ATC cells was confirmed by enzyme-linked immunosorbent assay (ELISA). Addition of SB431542, a TGFß receptor inhibitor, significantly decreased the Dectin1, CD163, SNAIL and SLUG expression stimulated by ATC cell-derived CM. We validated the clinical significance of the expression of TGFß ligands, their receptors, as well as SNAIL and SLUG in human ATC by analyzing public microarray datasets. We found that the expression of the main TGFß ligands, TGFß1 and TGFß3, along with their receptors, TGFR1 and TGFR2, as well as SLUG, was significantly higher in human ATC tissue samples than in normal thyroid tissues. Our findings indicate that ATC cell-secreted TGFß1 may play a key role in M2-like macrophage polarization of human monocytes and in the up-regulation of SNAIL and SLUG transcription factors. Thus, ours results uncovered a novel mechanism involved in the activation of TAMs by soluble factors released by ATC cells, which suggest potential therapeutic targets for ATC.
RESUMO
Hypothyroidism is commonly detected in patients with medulloblastoma (MB). A possible link between thyroid hormone (TH) signaling and MB pathogenicity has not been reported. Here, we find that TH plays a critical role in promoting tumor cell differentiation. Reduction in TH levels frees the TH receptor, TRα1, to bind to EZH2 and repress expression of NeuroD1, a transcription factor that drives tumor cell differentiation. Increased TH reverses EZH2-mediated repression of NeuroD1 by abrogating the binding of EZH2 and TRα1, thereby stimulating tumor cell differentiation and reducing MB growth. Importantly, TH-induced differentiation of tumor cells is not restricted by the molecular subgroup of MB. These findings establish an unprecedented association between TH signaling and MB pathogenicity, providing solid evidence for TH as a promising modality for MB treatment.
RESUMO
Hypothyroidism is commonly detected in patients with medulloblastoma (MB). However, whether thyroid hormone (TH) contributes to MB pathogenicity remains undetermined. Here, we find that TH plays a critical role in promoting tumor cell differentiation. Reduction in TH levels frees the TH receptor, TRα1, to bind to EZH2 and repress expression of NeuroD1, a transcription factor that drives tumor cell differentiation. Increased TH reverses EZH2-mediated repression of NeuroD1 by abrogating the binding of EZH2 and TRα1, thereby stimulating tumor cell differentiation and reducing MB growth. Importantly, TH-induced differentiation of tumor cells is not restricted by the molecular subgroup of MB, suggesting that TH can be used to broadly treat MB subgroups. These findings establish an unprecedented association between TH signaling and MB pathogenicity, providing solid evidence for TH as a promising modality for MB treatment.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular , Proteína Potenciadora do Homólogo 2 de Zeste , Meduloblastoma , Hormônios Tireóideos , Meduloblastoma/patologia , Meduloblastoma/metabolismo , Meduloblastoma/genética , Humanos , Diferenciação Celular/efeitos dos fármacos , Animais , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Camundongos , Hormônios Tireóideos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/tratamento farmacológico , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptores alfa dos Hormônios Tireóideos/metabolismo , Receptores alfa dos Hormônios Tireóideos/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Despite recent advances, understanding of molecular genetic alterations underlying thyroid carcinogenesis remains unclear. One key question is how dynamic temporal changes in global genomic expression affect carcinogenesis as the disease progresses. To address this question, we used a mouse model that spontaneously develops follicular thyroid cancer similar to human cancer (Thrb (PV/PV) mice). Using complementary DNA microarrays, we compared global gene expression profiles of thyroid tumors of Thrb (PV/PV) mice with the age- and gender-matched thyroids of wild-type mice at 3 weeks and at 2, 4, 6 and 14 months. These time points covered the pathological progression from early hyperplasia to capsular invasion, vascular invasion and eventual metastasis. Microarray data indicated that 462 genes were upregulated (Up-cluster genes) and 110 genes were downregulated (Down-cluster genes). Three major expression patterns (trending up, cyclical and spiking up and then down) and two (trending down and cyclical) were apparent in the Up-cluster and Down-cluster genes, respectively. Functional clustering of tumor-related genes followed by Ingenuity Pathways Analysis identified the transforming growth factor ß (TGF ß)-mediated network as key signaling pathways. Further functional analyses showed sustained activation of TGFß receptor-pSMAD2/3 signaling, leading to decreased expression of E-cadherin and increased expression of fibronectin, vimentin, collagens and laminins. These TGFß-induced changes facilitated epithelial-to-mesenchymal transition, which promotes cancer invasion and migration. Thus, complex temporal changes in gene expression patterns drive thyroid cancer progression, and persistent activation of TGFß-TGFRßII-pSMAD2/3 signaling leads to EMT, thus promoting metastasis. This study provides new understanding of progression and metastatic spread of human thyroid cancer.
Assuntos
Carcinogênese/metabolismo , Transdução de Sinais , Neoplasias da Glândula Tireoide/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Carcinogênese/genética , Análise por Conglomerados , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologiaRESUMO
Thyroid hormone (T(3)) acts in chondrocytes and bone-forming osteoblasts to control bone development and maintenance, but the signaling pathways mediating these effects are poorly understood. Thrb(PV/PV) mice have a severely impaired pituitary-thyroid axis and elevated thyroid hormone levels due to a dominant-negative mutant T(3) receptor (TRß(PV)) that cannot bind T(3) and interferes with the actions of wild-type TR. Thrb(PV/PV) mice have accelerated skeletal development due to unknown mechanisms. We performed microarray studies in primary osteoblasts from wild-type mice and Thrb(PV/PV) mice. Activation of the canonical Wnt signaling in Thrb(PV/PV) mice was confirmed by in situ hybridization analysis of Wnt target gene expression in bone during postnatal growth. By contrast, T(3) treatment inhibited Wnt signaling in osteoblastic cells, suggesting that T(3) inhibits the Wnt pathway by facilitating proteasomal degradation of ß-catenin and preventing its accumulation in the nucleus. Activation of the Wnt pathway in Thrb(PV/PV) mice, however, results from a gain of function for TRß(PV) that stabilizes ß-catenin despite the presence of increased thyroid hormone levels. These studies demonstrate novel interactions between T(3) and Wnt signaling pathways in the regulation of skeletal development and bone formation.
Assuntos
Mutação , Osteoblastos/metabolismo , Osteogênese/fisiologia , Hipófise/metabolismo , Receptores beta dos Hormônios Tireóideos/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Animais , Camundongos , Camundongos Mutantes , Osteoblastos/citologia , Estabilidade Proteica , Receptores beta dos Hormônios Tireóideos/genética , Tri-Iodotironina/metabolismo , Tri-Iodotironina/farmacologia , beta Catenina/genéticaRESUMO
Anaplastic thyroid cancer (ATC) is one of the most aggressive solid cancers in humans, with limited treatment options. Recent studies suggest that cancer stem cell (CSC) activity contributes to therapeutic resistance and recurrence of ATC. We show that the expression of the endogenous thyroid hormone receptor ß gene (THRB) is silenced in ATC and demonstrate that the exogenously expressed TRß suppresses CSC activity. Decitabine is one of the demethylation agents to treat myelodysplastic syndrome and acute myeloid leukemia patients and is currently in clinical trials for hematopoietic malignancies and solid tumors. We aim to show that the re-expression of the endogenous THRB gene by decitabine can attenuate CSC activity to block ATC tumor growth. We treated ATC cell lines derived from human ATC tumors (11T and 16T cells) with decitabine and evaluated the effects of the reactivated endogenous TRß on CSC activity in vitro and in vivo xenograft models. We found that treatment of 11T and 16T cells with decitabine reactivated the expression of endogenous TRß, as evidenced by western blot and immunohistochemical analyses. The expressed TRß inhibited cell proliferation by arresting cells at the S phase, increased apoptotic cell death by upregulation of cleaved caspase-3, and markedly suppressed the expression of CSC regulators, including cMYC, ALDH, SOX2, CD44, and ß-catenin. Decitabine also inhibited xenograft tumor growth by suppressing CSC activity, inhibiting cancer cell proliferation, and increasing apoptosis. Our findings suggest that re-expression of the endogenous TRß is a novel therapeutic approach for ATC via suppression of CSC activity.
Assuntos
Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Humanos , Carcinoma Anaplásico da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Receptores beta dos Hormônios Tireóideos/metabolismo , Genes erbA , Decitabina/metabolismo , Decitabina/farmacologia , Decitabina/uso terapêutico , Linhagem Celular Tumoral , Células-Tronco Neoplásicas/metabolismo , Apoptose , Proliferação de CélulasRESUMO
Background: Mutations of thyroid hormone receptor α (TRα1) result in resistance to thyroid hormone (RTHα), exhibiting symptoms of retarded growth, delayed bone maturation, anemia, and severe constipation. Using a mouse model of RTHα (Thra1PV/+ mouse), we aimed at understanding the molecular basis underlying the severe constipation observed in patients. Methods: The Thra1PV/+ mouse expresses a strong dominant negative mutant, PV, which has lost T3 binding and transcription activity. Thra1PV/+ mouse faithfully reproduces growth abnormalities and anemia as shown in RTHα patients and therefore is a valid model to examine causes of severe constipation in patients. We used histopathological analysis, confocal fluorescence imaging, transmission electron microscopy (TEM), and gene expression profiles to comprehensively analyze the colonic abnormalities of Thra1PV/+ mouse. Results: We found a significant increase in colonic transit time and decrease stool water content in Thra1PV/+ mouse, mimicking constipation as found in patients. Histopathological analysis showed expanded lamina propria filled with interstitium fluid between crypt columns, enlarged muscularis mucosa, and increased content of collagen in expanded submucosa. The TEM analysis revealed shorter muscle fibers with wider gap junctions between muscle cells, fewer caveolae, and hypoplastic interstitial cells of Cajal (ICC) in the rectal smooth muscles of Thra1PV/+ mice. These abnormal histological manifestations suggested defective intercellular transfer of small molecules, electrolytes, and signals for communication among muscles cells, validated by Lucifer Yellow transferring assays. Expression of key smooth muscle contractility regulators, such as calmodulin, myosin light-chain kinase, and phosphorylated myosin light chain, was markedly lower, and c-KIT signaling in ICC was attenuated, resulting in decreased contractility of the rectal smooth muscles of Thra1PV/+ mice. Collectively, these abnormal histopathological alterations and diminished contractility regulators led to the constipation exhibited in patients. Conclusions: This is the first demonstration that TRα1 mutants could act to cause abnormal rectum smooth muscle organization, defects in intercellular exchange of small molecules, and decreased expression of contractility regulators to weaken the contractility of rectal smooth muscles. These findings provide new insights into the molecular basis underlying constipation found in RTHα patients.
Assuntos
Anemia , Receptores alfa dos Hormônios Tireóideos , Humanos , Receptores alfa dos Hormônios Tireóideos/genética , Receptores alfa dos Hormônios Tireóideos/metabolismo , Hormônios Tireóideos , Mutação , Constipação Intestinal/genéticaRESUMO
Thyroid hormone receptor α1 (TRα1) mediates the genomic actions of thyroid hormone (T3). The biology of TRα1 in growth and development has been well studied, but the functional role of TRα1 in cancers remains to be elucidated. Analysis of the human thyroid cancer database of The Cancer Genome Atlas (TCGA) showed that THRA gene expression is lost in highly dedifferentiated anaplastic thyroid cancer (ATC). We, therefore, explored the effects of TRα1 on the progression of ATC. We stably expressed TRα1 in two human ATC cell lines, THJ-11T (11T-TRα1 #2, #7, and #8) and THJ-16T (16T-TRα1 #3, #4, and #8) cells. We found that the expressed TRα1 inhibited ATC cell proliferation and induced apoptosis. TCGA data showed that THRA gene expression was best correlated with the paired box gene 8 (PAX8). Consistently, we found that the PAX8 expression was barely detectable in parental 11T and 16T cells. However, PAX8 gene expression was elevated in 11T- and 16T-TRα1-expressing cells at the mRNA and protein levels. Using various molecular analyses, we found that TRα1 directly regulated the expression of the PAX8 gene. Single-cell transcriptomic analyses (scRNA-seq) demonstrated that TRα1 functions as a transcription factor through multiple signaling pathways to suppress tumor growth. Importantly, scRNA-seq analysis showed that TRα1-induced PAX8, via its transcription program, shifts the cell landscape of ATC toward a differentiated state. The present studies suggest that TRα1 is a newly identified regulator of thyroid differentiation and could be considered as a potential therapeutic target to improve the outcome of ATC patients.