RESUMEN
Anaplastic thyroid cancer (ATC) is a clinically aggressive form of undifferentiated thyroid cancer with limited treatment options. Immunotherapy for patients with ATC remains challenging. Tumor-associated macrophages (TAMs) constitute over 50% of ATC-infiltrating cells, and their presence is associated with a poor prognosis. Consequently, the development of new therapies targeting immune checkpoints in TAMs is considered a promising therapeutic approach for ATC. We have previously shown that soluble factors secreted by ATC cells induced pro-tumor M2-like polarization of human monocytes by upregulating the levels of the inhibitory receptor TIM3. Here, we extended our observations on ATC-cell-induced xenograft tumors. We observed a large number of immune cells infiltrating the ATC xenograft tumors. Significantly, 24-28% of CD45+ immune cells were macrophages (CD11b+ F4/80+). We further showed that 40% of macrophages were polarized toward a M2-like phenotype, as assessed by CD206 expression and by a significant increase in the Arg1/iNOS (M2/M1) ratio. Additionally, we found that ATC xenograft tumors had levels of TIM3 expression when determined by RT-PCR and immunofluorescence assays. Interestingly, we detected the expression of TIM3 in macrophages in ATC tumors by flow cytometry assays. Furthermore, TIM3 expression correlated with macrophage marker expression in human ATC. Our studies show that TIM3 is a newly identified immune checkpoint in macrophages. Since TIM3 is known as a negative immune regulator, it should be considered as a promising immunotherapeutic target for ATC.
RESUMEN
Congenital iodide transport defect is an uncommon autosomal recessive disorder caused by loss-of-function variants in the sodium iodide symporter (NIS)-coding SLC5A5 gene and leading to dyshormonogenic congenital hypothyroidism. Here, we conducted a targeted next-generation sequencing assessment of congenital hypothyroidism-causative genes in a cohort of nine unrelated pediatric patients suspected of having a congenital iodide transport defect based on the absence of 99mTc-pertechnetate accumulation in a eutopic thyroid gland. Although, unexpectedly, we could not detect pathogenic SLC5A5 gene variants, we identified two novel compound heterozygous TG gene variants (p.Q29* and c.177-2A>C), three novel heterozygous TG gene variants (p.F1542Vfs*20, p.Y2563C, and p.S523P), and a novel heterozygous DUOX2 gene variant (p.E1496Dfs*51). Splicing minigene reporter-based in vitro assays revealed that the variant c.177-2A>C affected normal TG pre-mRNA splicing, leading to the frameshift variant p.T59Sfs*17. The frameshift TG variants p.T59Sfs*17 and p.F1542Vfs*20, but not the DUOX2 variant p.E1496Dfs*51, were predicted to undergo nonsense-mediated decay. Moreover, functional in vitro expression assays revealed that the variant p.Y2563C reduced the secretion of the TG protein. Our investigation revealed unexpected findings regarding the genetics of congenital iodide transport defects, supporting the existence of yet to be discovered mechanisms involved in thyroid hormonogenesis.
Asunto(s)
Hipotiroidismo Congénito , Tiroglobulina , Niño , Hipotiroidismo Congénito/genética , Oxidasas Duales/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Yoduros/metabolismo , Mutación , Tiroglobulina/genéticaRESUMEN
Background: Congenital iodide transport defect (ITD) is an uncommon cause of dyshormonogenic congenital hypothyroidism characterized by the absence of active iodide accumulation in the thyroid gland. ITD is an autosomal recessive disorder caused by loss-of-function variants in the sodium/iodide symporter (NIS)-coding SLC5A5 gene. Objective: We aimed to identify, and if so to functionally characterize, novel ITD-causing SLC5A5 gene variants in a cohort of five unrelated pediatric patients diagnosed with dyshormonogenic congenital hypothyroidism with minimal to absent 99mTc-pertechnetate accumulation in the thyroid gland. Methods: The coding region of the SLC5A5 gene was sequenced using Sanger sequencing. In silico analysis and functional in vitro characterization of a novel synonymous variant were performed. Results: Sanger sequencing revealed a novel homozygous synonymous SLC5A5 gene variant (c.1326A>C in exon 11). In silico analysis revealed that the c.1326A>C variant is potentially deleterious for NIS pre-mRNA splicing. The c.1326A>C variant was predicted to lie within a putative exonic splicing enhancer reducing the binding of splicing regulatory trans-acting protein SRSF5. Splicing minigene reporter assay revealed that c.1326A>C causes exon 11 or exon 11 and 12 skipping during NIS pre-mRNA splicing leading to the NIS pathogenic variants p.G415_P443del and p.G415Lfs*32, respectively. Significantly, the frameshift variant p.G415Lfs*32 is predicted to be subjected to degradation by nonsense-mediated decay. Conclusions: We identified the first exonic synonymous SLC5A5 gene variant causing aberrant NIS pre-mRNA splicing, thus expanding the mutational landscape of the SLC5A5 gene leading to dyshormonogenic congenital hypothyroidism.
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Hipotiroidismo Congénito , Simportadores , Niño , Hipotiroidismo Congénito/genética , Exones , Humanos , Yoduros/metabolismo , Precursores del ARN , Simportadores/genéticaRESUMEN
Background: The sodium/iodide symporter (NIS) mediates active iodide accumulation in the thyroid follicular cell. Autosomal recessive iodide transport defect (ITD)-causing loss-of-function NIS variants lead to dyshormonogenic congenital hypothyroidism due to deficient iodide accumulation for thyroid hormonogenesis. Here, we aimed to identify, and if so to functionally characterize, novel ITD-causing NIS pathogenic variants in a patient diagnosed with severe dyshormonogenic congenital hypothyroidism due to a defect in iodide accumulation in the thyroid follicular cell, as suggested by nondetectable radioiodide accumulation in a normally located thyroid gland, as well as in salivary glands. Methods: The proposita NIS-coding SLC5A5 gene was sequenced using Sanger sequencing. In silico analysis and functional in vitro characterization of the novel NIS variants were performed. Results: Sanger sequencing revealed novel compound heterozygous SLC5A5 gene variants (c.970-3C>A and c.1106A>T, p.D369V). In silico analysis suggested that c.970-3C>A disrupts the canonical splice acceptor site located in intron 7. Splicing minigene reporter assay revealed that c.970-3C>A causes exon 8 skipping during NIS pre-mRNA splicing leading to the NIS pathogenic variant p.Y324Hfs*148. Moreover, in silico analysis indicated p.D369V as pathogenic. Functional in vitro studies demonstrated that p.D369V NIS does not mediate iodide accumulation, as p.D369V causes NIS to be retained in the endoplasmic reticulum. Mechanistically, we propose an intramolecular ionic interaction involving the ß carboxyl group of D369 and the guanidinium group of R130, located in transmembrane segment 4. Of note, an Asp residue at position 369-which is highly conserved in SLC5A family members-is required for functional NIS expression at the plasma membrane. Conclusions: We uncovered a critical intramolecular interaction between R130 and D369 required for NIS maturation and plasma membrane expression. Moreover, we identified the first intronic variant causing aberrant NIS pre-mRNA splicing, thus expanding the mutational landscape in the SLC5A5 gene leading to dyshormonogenic congenital hypothyroidism.
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Membrana Celular/efectos de los fármacos , Hipotiroidismo Congénito/tratamiento farmacológico , Simportadores/efectos de los fármacos , Membrana Celular/fisiología , Hipotiroidismo Congénito/genética , Hipotiroidismo Congénito/metabolismo , Humanos , Glándula Tiroides/metabolismoRESUMEN
Anaplastic thyroid cancer (ATC) is a highly aggressive type of thyroid cancer (TC). Currently, no effective target treatments are available that can improve overall survival, with ATC representing a major clinical challenge because of its remarkable lethality. Tumor-associated macrophages (TAMs) are the most evident cells in ATCs, and their high density is correlated with a poor prognosis. However, the mechanisms of how TAMs promote ATC progression remain poorly characterized. Here, we demonstrated that the treatment of human monocytes (THP-1 cells) with ATC cell-derived conditioned media (CM) promoted macrophage polarization, showing high levels of M2 markers. Furthermore, we found that STAT3 was activated, and this was correlated with an increased expression and secretion of the inflammatory cytokine interleukin-6. Remarkably, the M2-like macrophages obtained revealed tumor-promoting activity. A cytokine array analysis demonstrated that M2-like macrophage-derived CM contained high levels of TIM3, which is an important immune regulatory molecule. Consistently, TIM3 expression was up-regulated in THP-1 cells cultured with ATC cell-derived CM. Moreover, TIM3 blockade significantly reversed the polarization of THP-1 cells induced by ATC cell-secreted soluble factors. We validated the clinical significance of the TIM3 in human TC by analyzing public datasets and found that the expression of TIM3 and its ligand galectin 9 was significantly higher in human TC tissue samples than in normal thyroid tissues. Taken together, our findings identified a new mechanism by which TIM3 induces tumor-promoting M2-like macrophage polarization in TC. Furthermore, TIM3 interference might be a potential tool for treatment of patients with ATC.
RESUMEN
The sodium/iodide symporter (NIS) expresses at the basolateral plasma membrane of the thyroid follicular cell and mediates iodide accumulation required for normal thyroid hormonogenesis. Loss-of-function NIS variants cause congenital hypothyroidism due to impaired iodide accumulation in thyroid follicular cells underscoring the significance of NIS for thyroid physiology. Here we report novel findings derived from the thorough characterization of the nonsense NIS mutant p.R636* NIS-leading to a truncated protein missing the last eight amino acids-identified in twins with congenital hypothyroidism. R636* NIS is severely mislocalized into intracellular vesicular compartments due to the lack of a conserved carboxy-terminal type 1 PDZ-binding motif. As a result, R636* NIS is barely targeted to the plasma membrane and therefore iodide transport is reduced. Deletion of the PDZ-binding motif causes NIS accumulation into late endosomes and lysosomes. Using PDZ domain arrays, we revealed that the PDZ-domain containing protein SCRIB binds to the carboxy-terminus of NIS by a PDZ-PDZ interaction. Furthermore, in CRISPR/Cas9-based SCRIB deficient cells, NIS expression at the basolateral plasma membrane is compromised, leading to NIS localization into intracellular vesicular compartments. We conclude that the PDZ-binding motif is a plasma membrane retention signal that participates in the polarized expression of NIS by selectively interacting with the PDZ-domain containing protein SCRIB, thus retaining the transporter at the basolateral plasma membrane. Our data provide insights into the molecular mechanisms that regulate NIS expression at the plasma membrane, a topic of great interest in the thyroid cancer field considering the relevance of NIS-mediated radioactive iodide therapy for differentiated thyroid carcinoma.
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Proteínas de la Membrana/metabolismo , Simportadores/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Membrana Celular/metabolismo , Codón sin Sentido , Hipotiroidismo Congénito/genética , Hipotiroidismo Congénito/metabolismo , Secuencia Conservada , Perros , Endosomas/metabolismo , Células HEK293 , Humanos , Lisosomas/metabolismo , Células de Riñón Canino Madin Darby , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Dominios PDZ/genética , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Simportadores/química , Simportadores/genética , Glándula Tiroides/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genéticaRESUMEN
CONTEXT: Iodide transport defect (ITD) (Online Mendelian Inheritance in Man No. 274400) is an uncommon cause of dyshormonogenic congenital hypothyroidism due to loss-of-function variants in the SLC5A5 gene, which encodes the sodium/iodide symporter (NIS), causing deficient iodide accumulation in thyroid follicular cells. OBJECTIVE: This work aims to determine the molecular basis of a patient's ITD clinical phenotype. METHODS: The propositus was diagnosed with dyshormonogenic congenital hypothyroidism with minimal 99mTc-pertechnetate accumulation in a eutopic thyroid gland. The propositus SLC5A5 gene was sequenced. Functional in vitro characterization of the novel NIS variant was performed. RESULTS: Sanger sequencing revealed a novel homozygous missense p.G561E NIS variant. Mechanistically, the G561E substitution reduces iodide uptake, because targeting of G561E NIS to the plasma membrane is reduced. Biochemical analyses revealed that G561E impairs the recognition of an adjacent tryptophan-acidic motif by the kinesin-1 subunit kinesin light chain 2 (KLC2), interfering with NIS maturation beyond the endoplasmic reticulum, and reducing iodide accumulation. Structural bioinformatic analysis suggests that G561E shifts the equilibrium of the unstructured tryptophan-acidic motif toward a more structured conformation unrecognizable to KLC2. Consistently, knockdown of Klc2 causes defective NIS maturation and consequently decreases iodide accumulation in rat thyroid cells. Morpholino knockdown of klc2 reduces thyroid hormone synthesis in zebrafish larvae leading to a hypothyroid state as revealed by expression profiling of key genes related to the hypothalamic-pituitary-thyroid axis. CONCLUSION: We report a novel NIS pathogenic variant associated with dyshormonogenic congenital hypothyroidism. Detailed molecular characterization of G561E NIS uncovered the significance of KLC2 in thyroid physiology.
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Hipotiroidismo Congénito/genética , Errores Innatos del Metabolismo/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Simportadores/genética , Hormonas Tiroideas/metabolismo , Animales , Humanos , Recién Nacido , Yoduros/metabolismo , Cinesinas , Masculino , Mutación Missense , Fenotipo , Ratas , Glándula Tiroides/metabolismoRESUMEN
Background: The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor is a key regulator of cell survival, proliferation, and gene expression. Although activation of NF-κB signaling in thyroid follicular cells after thyrotropin (TSH) receptor (TSHR) engagement has been reported, the downstream signaling leading to NF-κB activation remains unexplored. Here, we sought to elucidate the mechanisms that regulate NF-κB signaling activation in response to TSH stimulation. Methods: Fisher rat-derived thyroid cell lines and primary cultures of NF-κB essential modulator (NEMO)-deficient mice thyrocytes were used as models. Signaling pathways leading to the activation of NF-κB were investigated by using chemical inhibitors and phospho-specific antibodies. Luciferase reporter gene assays and site-directed mutagenesis were used to monitor NF-κB-dependent gene transcriptional activity and the expression of thyroid differentiation markers was assessed by reverse transcription quantitative polymerase chain reaction and Western blot, respectively. Chromatin immunoprecipitation (ChIP) was carried out to investigate NF-κB subunit p65 DNA binding, and small interfering RNA (siRNA)-mediated gene knockdown approaches were used for studying gene function. Results: Using thyroid cell lines, we observed that TSH treatment leads to protein kinase C (PKC)-mediated canonical NF-κB p65 subunit nuclear expression. Moreover, TSH stimulation phosphorylated the kinase TAK-1, and its knockdown abolished TSH-induced NF-κB transcriptional activity. TSH induced the transcriptional activity of the NF-κB subunit p65 in a protein kinase A (PKA)-dependent phosphorylation at Ser-276. In addition, p65 phosphorylation at Ser-276 induced acetyl transferase p300 recruitment, leading to its acetylation on Lys-310 and thereby enhancing its transcriptional activity. Evaluation of the role played by NF-κB in thyroid physiology demonstrated that the canonical NF-κB inhibitor BAY 11-7082 reduced TSH-induced expression of thyroid differentiation markers. The involvement of NF-κB signaling in thyroid physiology was confirmed by assessing the TSH-induced gene expression in primary cultures of NEMO-deficient mice thyrocytes. ChIP and the knockdown experiments revealed that p65 is a nuclear effector of TSH actions, inducing the transcripcional expression of thyroid differentiation markers. Conclusions: Taken together, our results point to NF-κB being a pivotal mediator in the TSH-induced thyroid follicular cell differentiation, a relevant finding with potential physiological and pathophysiological implications.
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Diferenciación Celular/efectos de los fármacos , Glándula Tiroides/efectos de los fármacos , Tirotropina/farmacología , Factor de Transcripción ReIA/metabolismo , Acetilación , Animales , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones Noqueados , Fosforilación , Proteína Quinasa C/metabolismo , Ratas Endogámicas F344 , Transducción de Señal , Glándula Tiroides/metabolismo , Factor de Transcripción ReIA/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Iodide transport defect (ITD) is an autosomal recessive disorder caused by deficient iodide accumulation into the thyroid follicular cell. ITD is an uncommon cause of dyshormonogenetic congenital hypothyroidism that results from inactivating mutations in the sodium/iodide symporter (NIS)-coding SLC5A5 gene. NIS is a key basolateral plasma membrane glycoprotein that efficiently mediates active iodide uptake in the thyroid-constituting the first step in the biosynthesis of the iodine-containing thyroid hormones-and other tissues, including salivary glands, lactating breast, and small intestine. The proposita, a 20-day-old female born in 1992, was diagnosed with congenital hypothyroidism through newborn screening. ITD was suspected on the basis of nondetectable radioiodide accumulation in a normally located nongoitrous thyroid gland, as well as in salivary glands. Sanger sequencing revealed nonpreviously reported compound heterozygous missense SLC5A5 gene variants (c.991G>A, p.D331N and c.1.641C>A, p.S547R). Notably, these variants have not been reported in public databases (i.e., Exome Aggregation Consortium, 1000 Genomes, and Single Nucleotide Polymorphism). In silico analysis using prediction softwares (i.e., SIFT, Polyphen-2, and MutationTaster2) support the pathologic significance of p.D331N and p.S547R NIS. Moreover, functional in vitro studies demonstrate that D331N and S547R NIS severely reduce iodide uptake when the proteins are heterologously expressed in HEK-293T cells because of a pronounced impairment of D331N and S547R NIS targeting to the plasma membrane. Of note, a charged residue at position 331 and a serine residue at position 547-which are highly conserved in SLC5A family members-are required for NIS plasma membrane targeting. We report two novel missense pathogenic variants in a compound heterozygous state in the SLC5A5 gene, detected through Sanger sequencing, in a pediatric female patient with dyshormonogenic congenital hypothyroidism.
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Hipotiroidismo Congénito/genética , Simportadores/genética , Adolescente , Preescolar , Hipotiroidismo Congénito/sangre , Hipotiroidismo Congénito/tratamiento farmacológico , Femenino , Heterocigoto , Humanos , Recién Nacido , Tamizaje Neonatal , Tirotropina/sangre , Tiroxina/uso terapéuticoRESUMEN
Iodine is a crucial component of thyroid hormones; therefore, a key requirement for thyroid hormone biosynthesis is that iodide (I-) be actively accumulated in the thyroid follicular cell. The ability of the thyroid epithelia to concentrate I- is ultimately dependent on functional Na+/ I- symporter (NIS) expression at the plasma membrane. Underscoring the significance of NIS for thyroid physiology, loss-of-function mutations in the NIS-coding SLC5A5 gene cause an I- transport defect, resulting in dyshormonogenic congenital hypothyroidism. Moreover, I- accumulation in the thyroid cell constitutes the cornerstone for radioiodide ablation therapy for differentiated thyroid carcinoma. However, differentiated thyroid tumors often exhibit reduced (or even undetectable) I- transport compared with normal thyroid tissue, and they are diagnosed as cold nodules on thyroid scintigraphy. Paradoxically, immunohistochemistry analysis revealed that cold thyroid nodules do not express NIS or express normal, or even higher NIS levels compared with adjacent normal tissue, but NIS is frequently intracellularly retained, suggesting the presence of posttranslational abnormalities in the transport of the protein to the plasma membrane. Ultimately, a thorough comprehension of the mechanisms that regulate NIS transport to the plasma membrane would have multiple implications for radioiodide therapy, opening the possibility to identify new molecular targets to treat radioiodide-refractory thyroid tumors. Therefore, in this review, we discuss the current knowledge regarding posttranslational mechanisms that regulate NIS transport to the plasma membrane under physiological and pathological conditions affecting the thyroid follicular cell, a topic of great interest in the thyroid cancer field.
RESUMEN
The Na+/iodide (I-) symporter (NIS), a glycoprotein expressed at the basolateral plasma membrane of thyroid follicular cells, mediates I- accumulation for thyroid hormonogenesis and radioiodide therapy for differentiated thyroid carcinoma. However, differentiated thyroid tumors often exhibit lower I- transport than normal thyroid tissue (or even undetectable I- transport). Paradoxically, the majority of differentiated thyroid cancers show intracellular NIS expression, suggesting abnormal targeting to the plasma membrane. Therefore, a thorough understanding of the mechanisms that regulate NIS plasma membrane transport would have multiple implications for radioiodide therapy. In this study, we show that the intracellularly facing carboxy-terminus of NIS is required for the transport of the protein to the plasma membrane. Moreover, the carboxy-terminus contains dominant basolateral information. Using internal deletions and site-directed mutagenesis at the carboxy-terminus, we identified a highly conserved monoleucine-based sorting motif that determines NIS basolateral expression. Furthermore, in clathrin adaptor protein (AP)-1B-deficient cells, NIS sorting to the basolateral plasma membrane is compromised, causing the protein to also be expressed at the apical plasma membrane. Computer simulations suggest that the AP-1B subunit σ1 recognizes the monoleucine-based sorting motif in NIS carboxy-terminus. Although the mechanisms by which NIS is intracellularly retained in thyroid cancer remain elusive, our findings may open up avenues for identifying molecular targets that can be used to treat radioiodide-refractory thyroid tumors that express NIS intracellularly.