Publications by authors named "Pilar Santisteban"

72 Publications

Unraveling the Complex Interplay Between Transcription Factors and Signaling Molecules in Thyroid Differentiation and Function, From Embryos to Adults.

Front Endocrinol (Lausanne) 2021 20;12:654569. Epub 2021 Apr 20.

Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas (CSIC) y Universidad Autónoma de Madrid (UAM), Madrid, Spain.

Thyroid differentiation of progenitor cells occurs during embryonic development and in the adult thyroid gland, and the molecular bases of these complex and finely regulated processes are becoming ever more clear. In this , we describe the most recent advances in the study of transcription factors, signaling molecules and regulatory pathways controlling thyroid differentiation and development in the mammalian embryo. We also discuss the maintenance of the adult differentiated phenotype to ensure the biosynthesis of thyroid hormones. We will focus on endoderm-derived thyroid epithelial cells, which are responsible for the formation of the thyroid follicle, the functional unit of the thyroid gland. The use of animal models and pluripotent stem cells has greatly aided in providing clues to the complicated puzzle of thyroid development and function in adults. The so-called thyroid transcription factors - Nkx2-1, Foxe1, Pax8 and Hhex - were the first pieces of the puzzle identified in mice. Other transcription factors, either acting upstream of or directly with the thyroid transcription factors, were subsequently identified to, almost, complete the puzzle. Among them, the transcription factors Glis3, Sox9 and the cofactor of the Hippo pathway Taz, have emerged as important players in thyroid differentiation and development. The involvement of signaling molecules increases the complexity of the puzzle. In this context, the importance of Bmps, Fgfs and Shh signaling at the onset of development, and of TSH, IGF1 and TGFβ both at the end of terminal differentiation in embryos and in the adult thyroid, are well recognized. All of these aspects are covered herein. Thus, readers will be able to visualize the puzzle of thyroid differentiation with most - if not all - of the pieces in place.
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http://dx.doi.org/10.3389/fendo.2021.654569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8095082PMC
April 2021

A Positive Feedback Loop Between DICER1 and Differentiation Transcription Factors Is Important for Thyroid Tumorigenesis.

Thyroid 2021 Jun 9;31(6):912-921. Epub 2020 Dec 9.

Instituto de Investigaciones Biomédicas "Alberto Sols," Consejo Superior Investigaciones Científicas, and Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain.

DICER1 plays a central role in microRNA biogenesis and functions as a tumor suppressor in thyroid cancer, which is the most frequent endocrine malignancy with a rapidly increasing incidence. Thyroid cancer progression is associated with loss of cell differentiation and reduced expression of thyroid differentiation genes and response to thyrotropin (TSH). Here we investigated whether a molecular link exists between DICER1 and thyroid differentiation pathways. We used bioinformatic tools to search for transcription factor binding sites in the promoter. DICER1, NKX2-1, PAX8, and CREB expression levels were evaluated by gene and protein expression and by interrogation of The Cancer Genome Atlas (TCGA) thyroid cancer data. Transcription factor binding and activity were assayed by chromatin immunoprecipitation, band-shift analysis, and promoter-reporter gene activity. Gene-silencing and overexpression approaches were used to elucidate the functional link between DICER1 and differentiation. We identified binding sites for NKX2-1 and CREB within the promoter and found that both transcription factors are functional in thyroid cells. TSH induced expression in differentiated thyroid cells, at least in part, through the cAMP/PKA/CREB pathway. TCGA analysis revealed a significant positive correlation between CREB and DICER1 expression in human thyroid tumors. NKX2-1 overexpression increased promoter activity and expression , and this was significantly greater in the presence of CREB and/or PAX8. Gain- and loss-of-function assays revealed that DICER1 regulates NKX2-1 expression in thyroid tumor cells and , thus establishing a positive feedback loop between both proteins. We also found a positive correlation between NKX2-1 and DICER1 expression in human thyroid tumors. DICER1 silencing decreased PAX8 expression and, importantly, the expression and activity of the sodium iodide symporter, which is essential for the diagnostic and therapeutic use of radioiodine in thyroid cancer. The differentiation transcription factors NKX2.1, PAX8, and CREB act in a positive feedback loop with DICER1. As the expression of these transcription factors is markedly diminished in thyroid cancer, our findings suggest that DICER1 downregulation in this cancer is mediated, at least partly, through impairment of its transcription.
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http://dx.doi.org/10.1089/thy.2020.0297DOI Listing
June 2021

RAS Subcellular Localization Inversely Regulates Thyroid Tumor Growth and Dissemination.

Cancers (Basel) 2020 Sep 10;12(9). Epub 2020 Sep 10.

Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Consejo Superior de Investigaciones Científicas (CSIC)-Universidad de Cantabria. Santander, E-39011 Cantabria, Spain.

mutations are the second most common genetic alteration in thyroid tumors. However, the extent to which they are associated with the most aggressive phenotypes is still controversial. Regarding their malignancy, the majority of mutant tumors are classified as undetermined, which complicates their clinical management and can lead to undesired under- or overtreatment. Using the chick embryo spontaneous metastasis model, we herein demonstrate that the aggressiveness of HRAS-transformed thyroid cells, as determined by the ability to extravasate and metastasize at distant organs, is orchestrated by HRAS subcellular localization. Remarkably, aggressiveness inversely correlates with tumor size. In this respect, we also show that RAS site-specific capacity to regulate tumor growth and dissemination is dependent on VEGF-B secretion. Furthermore, we have identified the acyl protein thioesterase APT-1 as a determinant of thyroid tumor growth versus dissemination. We show that alterations in APT-1 expression levels can dramatically affect the behavior of thyroid tumors, based on its role as a regulator of HRAS sublocalization at distinct plasma membrane microdomains. In agreement, APT-1 emerges in thyroid cancer clinical samples as a prognostic factor. As such, APT-1 levels could serve as a biomarker that could help in the stratification of mutant thyroid tumors based on their aggressiveness.
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http://dx.doi.org/10.3390/cancers12092588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565207PMC
September 2020

A Novel Role for the Tumor Suppressor Gene in Tumorigenesis and Chemotherapy Response.

Cancers (Basel) 2020 03 26;12(4). Epub 2020 Mar 26.

Epigenetics Laboratory, INGEMM, Hospital La PAZ, 28046 Madrid, Spain.

Despite often leading to platinum resistance, platinum-based chemotherapy continues to be the standard treatment for many epithelial tumors. In this study we analyzed and validated the cytogenetic alterations that arise after treatment in four lung and ovarian paired cisplatin-sensitive/resistant cell lines by 1-million microarray-based comparative genomic hybridization (array-CGH) and qRT-PCR methodologies. RNA-sequencing, functional transfection assays, and gene-pathway activity analysis were used to identify genes with a potential role in the development of this malignancy. The results were further explored in 55 lung and ovarian primary tumors and control samples, and in two extensive in silico databases. Long-term cell exposure to platinum induces the frequent deletion of gene. Its expression re-sensitized tumor cells to platinum and recovered the levels of Wnt/β-catenin transcriptional activity. expression was also frequently downregulated in epithelial tumors, predicting a worse overall survival. We also identified an inverse correlation between and expression, revealing that Non-small cell lung cancer (NSCLC) patients with lower expression of had a better overall survival rate. We defined the implication of as a molecular mechanism behind the development of cisplatin resistance probably through the activation of the Wnt-signaling pathway. This data highlights the possible role of and as novel therapeutic targets for platinum resistant tumors.
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http://dx.doi.org/10.3390/cancers12040786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226299PMC
March 2020

Gene network transitions in embryos depend upon interactions between a pioneer transcription factor and core histones.

Nat Genet 2020 04 16;52(4):418-427. Epub 2020 Mar 16.

Institute for Regenerative Medicine, Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Gene network transitions in embryos and other fate-changing contexts involve combinations of transcription factors. A subset of fate-changing transcription factors act as pioneers; they scan and target nucleosomal DNA and initiate cooperative events that can open the local chromatin. However, a gap has remained in understanding how molecular interactions with the nucleosome contribute to the chromatin-opening phenomenon. Here we identified a short α-helical region, conserved among FOXA pioneer factors, that interacts with core histones and contributes to chromatin opening in vitro. The same domain is involved in chromatin opening in early mouse embryos for normal development. Thus, local opening of chromatin by interactions between pioneer factors and core histones promotes genetic programming.
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http://dx.doi.org/10.1038/s41588-020-0591-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901023PMC
April 2020

ADAR1-mediated RNA editing is a novel oncogenic process in thyroid cancer and regulates miR-200 activity.

Oncogene 2020 04 10;39(18):3738-3753. Epub 2020 Mar 10.

Instituto de Investigaciones Biomédicas "Alberto Sols"; Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid (UAM), Madrid, Spain.

Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. A-to-I editing of RNA is a widespread posttranscriptional process that has recently emerged as an important mechanism in cancer biology. A-to-I editing levels are high in several human cancers, including thyroid cancer, but ADAR1 editase-dependent mechanisms governing thyroid cancer progression are unexplored. To address the importance of RNA A-to-I editing in thyroid cancer, we examined the role of ADAR1. Loss-of-function analysis showed that ADAR1 suppression profoundly repressed proliferation, invasion, and migration in thyroid tumor cell models. These observations were validated in an in vivo xenograft model, which showed that ADAR1-silenced cells had a diminished ability to form tumors. RNA editing of miRNAs has the potential to markedly alter target recognition. According to TCGA data, the tumor suppressor miR-200b is overedited in thyroid tumors, and its levels of editing correlate with a worse progression-free survival and disease stage. We confirmed miR-200b overediting in thyroid tumors and we showed that edited miR-200b has weakened activity against its target gene ZEB1 in thyroid cancer cells, likely explaining the reduced aggressiveness of ADAR1-silenced cells. We also found that RAS, but not BRAF, modulates ADAR1 levels, an effect mediated predominantly by PI3K and in part by MAPK. Lastly, pharmacological inhibition of ADAR1 activity with the editing inhibitor 8-azaadenosine reduced cancer cell aggressiveness. Overall, our data implicate ADAR1-mediated A-to-I editing as an important pathway in thyroid cancer progression, and highlight RNA editing as a potential therapeutic target in thyroid cancer.
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http://dx.doi.org/10.1038/s41388-020-1248-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190574PMC
April 2020

FOXE1 regulates migration and invasion in thyroid cancer cells and targets ZEB1.

Endocr Relat Cancer 2020 03;27(3):137-151

Instituto de Investigaciones Biomédicas 'Alberto Sols', Consejo Superior Investigaciones Científicas, and Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain.

FOXE1 is a thyroid-specific transcription factor essential for thyroid gland development and maintenance of the differentiated state. Interestingly, a strong association has been recently described between FOXE1 expression and susceptibility to thyroid cancer, but little is known about the mechanisms underlying FOXE1-induced thyroid tumorigenesis. Here, we used a panel of human thyroid cancer-derived cell lines covering the spectrum of thyroid cancer phenotypes to examine FOXE1 expression and to test for correlations between FOXE1 expression, the allele frequency of two SNPs and a length polymorphism in or near the FOXE1 locus associated with cancer susceptibility, and the migration ability of thyroid cancer cell lines. Results showed that FOXE1 expression correlated with differentiation status according to histological sub-type, but not with SNP genotype or cell migration ability. However, loss-and-gain-of-function experiments revealed that FOXE1 modulates cell migration, suggesting a role in epithelial-to-mesenchymal transition (EMT). Our previous genome-wide expression analysis identified Zeb1, a major EMT inducer, as a putative Foxe1 target gene. Indeed, gene silencing of FOXE1 decreased ZEB1 expression, whereas its overexpression increased ZEB1 transcriptional activity. FOXE1 was found to directly interact with the ZEB1 promoter. Lastly, ZEB1 silencing decreased the ability of thyroid tumoral cells to migrate and invade, pointing to its importance in thyroid tumor mestastases. In conclusion, we have identified ZEB1 as a bona fide target of FOXE1 in thyroid cancer cells, which provides new insights into the role of FOXE1 in regulating cell migration and invasion in thyroid cancer.
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http://dx.doi.org/10.1530/ERC-19-0156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993207PMC
March 2020

Impaired Gene Expression Due to Iodine Excess in the Development and Differentiation of Endoderm and Thyroid Is Associated with Epigenetic Changes.

Thyroid 2020 04 23;30(4):609-620. Epub 2020 Jan 23.

Instituto de Investigaciones Biomédicas "Alberto Sols," CSIC-UAM, Madrid, Spain.

Thyroid hormone (TH) synthesis is essential for the control of development, growth, and metabolism in vertebrates and depends on a sufficient dietary iodine intake. Importantly, both iodine deficiency and iodine excess (IE) impair TH synthesis, causing serious health problems especially during fetal/neonatal development. While it is known that IE disrupts thyroid function by inhibiting thyroid gene expression, its effects on thyroid development are less clear. Accordingly, this study sought to investigate the effects of IE during the embryonic development/differentiation of endoderm and the thyroid gland. We used the murine embryonic stem (ES) cell model of directed differentiation to assess the impact of IE on the generation of endoderm and thyroid cells. Additionally, we subjected endoderm and thyroid explants obtained during early gestation to IE and evaluated gene and protein expression of endodermal markers in both models. ES cells were successfully differentiated into endoderm cells and, subsequently, into thyrocytes expressing the specific thyroid markers , , , and . IE exposure decreased the messenger RNA (mRNA) levels of the main endoderm markers , , , , and in both ES cell-derived endoderm cells and embryonic explants. Interestingly, IE also decreased the expression of the main thyroid markers in ES cell-derived thyrocytes and thyroid explants. Finally, we demonstrate that DNA methyltransferase expression was increased by exposure to IE, and this was accompanied by hypermethylation and hypoacetylation of histone H3, pointing to an association between the gene repression triggered by IE and the observed epigenetic changes. These data establish that IE treatment is deleterious for embryonic endoderm and thyroid gene expression.
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http://dx.doi.org/10.1089/thy.2018.0658DOI Listing
April 2020

BRAF V600E status may facilitate decision-making on active surveillance of low-risk papillary thyroid microcarcinoma.

Eur J Cancer 2020 01 29;124:161-169. Epub 2019 Nov 29.

Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA. Electronic address:

Introduction: Conservative active surveillance has been proposed for low-risk papillary thyroid microcarcinoma (PTMC), defined as ≤1.0 cm and lacking clinical aggressive features, but controversy exists with accepting it as not all such PTMCs are uniformly destined for benign prognosis. This study investigated whether BRAF V600E status could further risk stratify PTMC, particularly low-risk PTMC, and can thus help with more accurate case selection for conservative management.

Methods: This international multicenter study included 743 patients treated with total thyroidectomy for PTMC (584 women and 159 men), with a median age of 49 years (interquartile range [IQR], 39-59 years) and a median follow-up time of 53 months (IQR, 25-93 months).

Results: On overall analyses of all PTMCs, tumour recurrences were 6.4% (32/502) versus 10.8% (26/241) in BRAF mutation-negative versus BRAF mutation-positive patients (P = 0.041), with a hazard ratio (HR) of 2.44 (95% CI (confidence interval), 1.15-5.20) after multivariate adjustment for confounding clinical factors. On the analyses of low-risk PTMC, recurrences were 1.3% (5/383) versus 4.3% (6/139) in BRAF mutation-negative versus BRAF mutation-positive patients, with an HR of 6.65 (95% CI, 1.80-24.65) after adjustment for confounding clinical factors. BRAF mutation was associated with a significant decline in the Kaplan-Meier recurrence-free survival curve in low-risk PTMC.

Conclusions: BRAF V600E differentiates the recurrence risk of PTMC, particularly low-risk PTMC. Given the robust negative predictive value, conservative active surveillance of BRAF mutation-negative low-risk PTMC is reasonable whereas the increased recurrence risk and other well-known adverse effects of BRAF V600E make the feasibility of long-term conservative surveillance uncertain for BRAF mutation-positive PTMC.
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http://dx.doi.org/10.1016/j.ejca.2019.10.017DOI Listing
January 2020

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice.

J Vis Exp 2019 08 23(150). Epub 2019 Aug 23.

Instituto de Investigaciones Biomédicas, CSIC-UAM; Ciberonc, Instituto de Salud Carlos III;

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.
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http://dx.doi.org/10.3791/59322DOI Listing
August 2019

miRNA-Directed Regulation of the Main Signaling Pathways in Thyroid Cancer.

Front Endocrinol (Lausanne) 2019 2;10:430. Epub 2019 Jul 2.

Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior Investigaciones Científicas and Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain.

In the last two decades, great strides have been made in the study of microRNAs in development and in diseases such as cancer, as reflected in the exponential increase in the number of reviews on this topic including those on undifferentiated and well-differentiated thyroid cancer. Nevertheless, few reviews have focused on understanding the functional significance of the most up- or down-regulated miRNAs in thyroid cancer for the main signaling pathways hyperactivated in this tumor type. The aim of this review is to discuss the major miRNAs targeting proteins of the MAPK, PI3K, and TGFβ pathways, to define their mechanisms of action through the 3'UTR regions of their target genes, and to describe how they affect thyroid tumorigenesis through their actions on cell proliferation, migration, and invasion. Given the importance of miRNAs in cancer as diagnostic, prognostic and therapeutic candidates, a better understanding of this cross-talk might shed new light on the biomedical treatment of thyroid cancer.
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http://dx.doi.org/10.3389/fendo.2019.00430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614345PMC
July 2019

Regulators of the RAS-ERK pathway as therapeutic targets in thyroid cancer.

Endocr Relat Cancer 2019 06;26(6):R319-R344

Instituto de Investigaciones Biomédicas 'Alberto Sols', Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid (UAM), Madrid, Spain.

Thyroid cancer is mostly an ERK-driven carcinoma, as up to 70% of thyroid carcinomas are caused by mutations that activate the RAS/ERK mitogenic signaling pathway. The incidence of thyroid cancer has been steadily increasing for the last four decades; yet, there is still no effective treatment for advanced thyroid carcinomas. Current research efforts are focused on impairing ERK signaling with small-molecule inhibitors, mainly at the level of BRAF and MEK. However, despite initial promising results in animal models, the clinical success of these inhibitors has been limited by the emergence of tumor resistance and relapse. The RAS/ERK pathway is an extremely complex signaling cascade with multiple points of control, offering many potential therapeutic targets: from the modulatory proteins regulating the activation state of RAS proteins to the scaffolding proteins of the pathway that provide spatial specificity to the signals, and finally, the negative feedbacks and phosphatases responsible for inactivating the pathway. The aim of this review is to give an overview of the biology of RAS/ERK regulators in human cancer highlighting relevant information on thyroid cancer and future areas of research.
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http://dx.doi.org/10.1530/ERC-19-0098DOI Listing
June 2019

Impaired microRNA processing by DICER1 downregulation endows thyroid cancer with increased aggressiveness.

Oncogene 2019 07 9;38(27):5486-5499. Epub 2019 Apr 9.

Instituto de Investigaciones Biomédicas "Alberto Sols"; Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid (UAM), Madrid, Spain.

The global downregulation of microRNAs (miRNAs) is emerging as a common hallmark of cancer. However, the mechanisms underlying this phenomenon are not well known. We identified that the oncogenic miR-146b-5p attenuates miRNA biosynthesis by targeting DICER1 and reducing its expression. DICER1 overexpression inhibited all the miR-146b-induced aggressive phenotypes in thyroid cells. Systemic injection of an anti-miR-146b in mice with orthotopic thyroid tumors suppressed tumor growth and recovered DICER1 levels. Notably, DICER1 downregulation promoted proliferation, migration, invasion, and epithelial-mesenchymal transition through miRNA downregulation. Our analysis of The Cancer Genome Atlas revealed a general decrease in DICER1 expression in thyroid cancer that was associated with a worse clinical outcome. Administration of the small-molecule enoxacin to promote DICER1 complex activity reduced tumor aggressiveness both in vitro and in vivo. Overall, our data confirm DICER1 as a tumor suppressor and show that oncogenic miR-146b contributes to its downregulation. Moreover, our results highlight a potential therapeutic application of RNA-based therapies including miRNA inhibitors and restoration of the biogenesis machinery, which may provide treatments for thyroid and other cancers.
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http://dx.doi.org/10.1038/s41388-019-0804-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6755984PMC
July 2019

Regulation of Foxe1 by Thyrotropin and Transforming Growth Factor Beta Depends on the Interplay Between Thyroid-Specific, CREB and SMAD Transcription Factors.

Thyroid 2019 05 13;29(5):714-725. Epub 2019 Feb 13.

1 Instituto de Investigaciones Biomédicas "Alberto Sols," Consejo Superior de Investigaciones Científicas (CSIC) y Universidad Autónoma de Madrid (UAM), Madrid, Spain.

Thyroid follicular cells are characterized by the expression of a specific set of genes necessary for the synthesis and secretion of thyroid hormones, which are in turn regulated by the transcription factors Nkx2-1, Pax8, and Foxe1. Thyroid differentiation is finely tuned by the balance between positive regulatory signals, including thyrotropin (TSH), and by negative regulatory signals, such as transforming growth factor beta (TGF-β), which counteracts the action of TSH. A role for Foxe1 as a mediator of hormonal and growth-factor control of thyroid differentiation has been previously suggested. Therefore, the aim of this work was to study the mechanisms governing Foxe1 expression to define the ligands and signals that regulate one of the important factors in thyroid differentiation. Expression of Foxe1 was evaluated in rat PCCl3 thyroid follicular cells under different treatments. The mouse promoter was cloned, and site-directed mutagenesis was undertaken to study its transcriptional regulation and to identify response elements. Protein/DNA binding assays were performed to evaluate the binding of different transcription factors, and gene-silencing approaches were used to elucidate their functional roles. analysis of the promoter identified binding sites for Nkx2-1, Pax8, Foxe1, and Smad proteins, as well as cAMP-response element (CRE) sites. It was found that both CRE-binding protein and CRE modulator were necessary for the TSH-mediated induction of Foxe1 expression via the cAMP/PKA signaling pathway. Moreover, transcription of was regulated by Nkx2-1 and Pax8 and by itself, suggesting an autoregulatory mechanism of activation and an important role for thyroid transcription factors. Finally, TGF-β, through Smad proteins, inhibited the TSH-induced Foxe1 expression. This study shows that Foxe1 is the final target of TSH/cAMP and TGF-β regulation that mediates expression of thyroid differentiation genes, and provides evidence of an interplay between CRE-binding proteins, thyroid transcription factors, and Smad proteins in its regulation. Thus, Foxe1 plays an important role in the complex transcriptional network that regulates thyroid follicular cell differentiation.
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http://dx.doi.org/10.1089/thy.2018.0136DOI Listing
May 2019

mRNA circularization by METTL3-eIF3h enhances translation and promotes oncogenesis.

Nature 2018 09 19;561(7724):556-560. Epub 2018 Sep 19.

Stem Cell Program, Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA, USA.

N-methyladenosine (mA) modification of mRNA is emerging as an important regulator of gene expression that affects different developmental and biological processes, and altered mA homeostasis is linked to cancer. mA modification is catalysed by METTL3 and enriched in the 3' untranslated region of a large subset of mRNAs at sites close to the stop codon. METTL3 can promote translation but the mechanism and relevance of this process remain unknown. 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 mA-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.
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http://dx.doi.org/10.1038/s41586-018-0538-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6234840PMC
September 2018

BRAF V600E Confers Male Sex Disease-Specific Mortality Risk in Patients With Papillary Thyroid Cancer.

J Clin Oncol 2018 09 2;36(27):2787-2795. Epub 2018 Aug 2.

Fei Wang, Shihua Zhao, and Yangang Wang, The Affiliated Hospital of Qingdao University, Qingdao, People's Republic of China; Fei Wang, Xiaopei Shen, Guangwu Zhu, Rengyun Liu, and Mingzhao Xing, Johns Hopkins University School of Medicine, Baltimore, MD; David Viola and Rossella Elisei, University of Pisa, Pisa; Efisio Puxeddu, University of Perugia, Perugia; Laura Fugazzola and Carla Colombo, Istituto Auxologico Italiano, Istituto di Recovero e Cura a Carattere Scientifico (IRCCS), and University of Milan, Milan; Caterina Mian, University of Padua; Federica Vianello, Veneto Institute of Oncology, IRCCS, Padua, Italy; Barbara Jarzab and Agnieszka Czarniecka, Maria Sklodowska-Curie Institute Oncology Center, Gliwice, Poland; Alfred K. Lam, Griffith University, Gold Coast, Queensland; Christine J. O'Neill, Mark S. Sywak, and Roderick Clifton-Bligh, University of Sydney, Sydney, New South Wales, Australia; Linwah Yip, University of Pittsburgh, Pittsburgh, PA; Garcilaso Riesco-Eizaguirre, Hospital Universitario La Paz and Hospital Universitario de Móstoles; Garcilaso Riesco-Eizaguirre and Pilar Santisteban, Biomedical Research Institute "Alberto Sols" and Health Institute Carlos III, Madrid, Spain; and Bela Bendlova and Vlasta Sýkorová, Institute of Endocrinology, Prague, Czech Republic.

Purpose To test whether the prognostic risk of male sex in papillary thyroid cancer (PTC) is determined by BRAF V600E and can thus be stratified by BRAF status. Patients and Methods We retrospectively investigated the relationship between male sex and clinicopathologic outcomes in PTC, particularly mortality, with respect to BRAF status in 2,638 patients (male, n = 623; female, n = 2,015) from 11 centers in six countries, with median age of 46 years (interquartile range, 35-58 years) at diagnosis and median follow-up time of 58 months (interquartile range, 26-107 months). Results Distant metastasis rates in men and women were not different in wild-type BRAF PTC but were different in BRAF V600E PTC: 8.9% (24 of 270) and 3.7% (30 of 817; P = .001), respectively. In wild-type BRAF PTC, mortality rates were 1.4% (five of 349) versus 0.9% (11 of 1175) in men versus women ( P = .384), with a hazard ratio (HR) of 1.59 (95% CI, 0.55 to 4.57), which remained insignificant at 0.70 (95% CI, 0.23 to 2.09) after clinicopathologic multivariable adjustment. In BRAF V600E PTC, mortality rates were 6.6% (18 of 272) versus 2.9% (24 of 822) in men versus women ( P = .006), with an HR of 2.43 (95% CI, 1.30 to 4.53), which remained significant at 2.74 (95% CI, 1.38 to 5.43) after multivariable adjustment. In conventional-variant PTC, male sex similarly had no effect in wild-type BRAF patients; mortality rates in BRAF V600E patients were 7.2% (16 of 221) versus 2.9% (19 of 662) in men versus women ( P = .004), with an HR of 2.86 (95% CI, 1.45 to 5.67), which remained significant at 3.51 (95% CI, 1.62 to 7.63) after multivariable adjustment. Conclusion Male sex is a robust independent risk factor for PTC-specific mortality in BRAF V600E patients but not in wild-type BRAF patients. The prognostic risk of male sex in PTC can thus be stratified by BRAF status in clinical application.
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http://dx.doi.org/10.1200/JCO.2018.78.5097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145834PMC
September 2018

An oncogenic role for microRNA-146b in the thyroid.

Oncoscience 2018 May 27;5(5-6):155-156. Epub 2018 Jun 27.

Instituto de Investigaciones Biomédicas, CSIC-UAM, Ciberonc, Madrid, Spain.

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http://dx.doi.org/10.18632/oncoscience.432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6049317PMC
May 2018

NFE2-Related Transcription Factor 2 Coordinates Antioxidant Defense with Thyroglobulin Production and Iodination in the Thyroid Gland.

Thyroid 2018 06;28(6):780-798

1 Service of Endocrinology, Diabetology and Metabolism, Lausanne University Hospital , Lausanne, Switzerland .

Background: The thyroid gland has a special relationship with oxidative stress. While generation of oxidative substances is part of normal iodide metabolism during thyroid hormone synthesis, the gland must also defend itself against excessive oxidation in order to maintain normal function. Antioxidant and detoxification enzymes aid thyroid cells to maintain homeostasis by ameliorating oxidative insults, including during exposure to excess iodide, but the factors that coordinate their expression with the cellular redox status are not known. The antioxidant response system comprising the ubiquitously expressed NFE2-related transcription factor 2 (Nrf2) and its redox-sensitive cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (Keap1) defends tissues against oxidative stress, thereby protecting against pathologies that relate to DNA, protein, and/or lipid oxidative damage. Thus, it was hypothesized that Nrf2 should also have important roles in maintaining thyroid homeostasis.

Methods: Ubiquitous and thyroid-specific male C57BL6J Nrf2 knockout (Nrf2-KO) mice were studied. Plasma and thyroids were harvested for evaluation of thyroid function tests by radioimmunoassays and of gene and protein expression by real-time polymerase chain reaction and immunoblotting, respectively. Nrf2-KO and Keap1-KO clones of the PCCL3 rat thyroid follicular cell line were generated using CRISPR/Cas9 technology and were used for gene and protein expression studies. Software-predicted Nrf2 binding sites on the thyroglobulin enhancer were validated by site-directed in vitro mutagenesis and chromatin immunoprecipitation.

Results: The study shows that Nrf2 mediates antioxidant transcriptional responses in thyroid cells and protects the thyroid from oxidation induced by iodide overload. Surprisingly, it was also found that Nrf2 has a dramatic impact on both the basal abundance and the thyrotropin-inducible intrathyroidal abundance of thyroglobulin (Tg), the precursor protein of thyroid hormones. This effect is mediated by cell-autonomous regulation of Tg gene expression by Nrf2 via its direct binding to two evolutionarily conserved antioxidant response elements in an upstream enhancer. Yet, despite upregulating Tg levels, Nrf2 limits Tg iodination both under basal conditions and in response to excess iodide.

Conclusions: Nrf2 exerts pleiotropic roles in the thyroid gland to couple cell stress defense mechanisms to iodide metabolism and the thyroid hormone synthesis machinery, both under basal conditions and in response to excess iodide.
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http://dx.doi.org/10.1089/thy.2018.0018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5994681PMC
June 2018

Functional Toll-like Receptor 4 Overexpression in Papillary Thyroid Cancer by MAPK/ERK-Induced ETS1 Transcriptional Activity.

Mol Cancer Res 2018 05 9;16(5):833-845. Epub 2018 Mar 9.

Centro de Investigaciones en Bioquímica Clínica e Inmunología - Consejo Nacional de Investigaciones Científicas y Técnicas (CIBICI-CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

Emerging evidence suggests that unregulated Toll-like receptor (TLR) signaling promotes tumor survival signals, thus favoring tumor progression. Here, the mechanism underlying TLR4 overexpression in papillary thyroid carcinomas (PTC) mainly harboring the BRAF mutation was studied. TLR4 was overexpressed in PTC compared with nonneoplastic thyroid tissue. Moreover, paired clinical specimens of primary PTC and its lymph node metastasis showed a significant upregulation of TLR4 levels in the metastatic tissues. In agreement, conditional BRAF expression in normal rat thyroid cells and mouse thyroid tissue upregulated TLR4 expression levels. Furthermore, functional TLR4 expression was demonstrated in PTC cells by increased NF-κB transcriptional activity in response to the exogenous TLR4-agonist lipopolysaccharide. Of note, The Cancer Genome Atlas data analysis revealed that BRAF-positive tumors with high TLR4 expression were associated with shorter disease-free survival. Transcriptomic data analysis indicated a positive correlation between TLR4 expression levels and MAPK/ERK signaling activation. Consistently, chemical blockade of MAPK/ERK signaling abrogated BRAF-induced TLR4 expression. A detailed study of the promoter revealed a critical MAPK/ERK-sensitive Ets-binding site involved in BRAF responsiveness. Subsequent investigation revealed that the Ets-binding factor ETS1 is critical for BRAF-induced MAPK/ERK signaling-dependent gene expression. Together, these data indicate that functional TLR4 overexpression in PTCs is a consequence of thyroid tumor-oncogenic driver dysregulation of MAPK/ERK/ETS1 signaling. Considering the participation of aberrant NF-κB signaling activation in the promotion of thyroid tumor growth and the association of high TLR4 expression with more aggressive tumors, this study suggests a prooncogenic potential of TLR4 downstream signaling in thyroid tumorigenesis. .
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http://dx.doi.org/10.1158/1541-7786.MCR-17-0433DOI Listing
May 2018

Role of iodide metabolism in physiology and cancer.

Endocr Relat Cancer 2018 04 1;25(4):R225-R245. Epub 2018 Feb 1.

CiberOnc, Instituto de Salud Carlos III, Madrid, Spain.

Iodide (I) metabolism is crucial for the synthesis of thyroid hormones (THs) in the thyroid and the subsequent action of these hormones in the organism. I is principally transported by the sodium iodide symporter (NIS) and by the anion exchanger PENDRIN, and recent studies have demonstrated the direct participation of new transporters including anoctamin 1 (ANO1), cystic fibrosis transmembrane conductance regulator (CFTR) and sodium multivitamin transporter (SMVT). Several of these transporters have been found expressed in various tissues, implicating them in I recycling. New research supports the exciting idea that I participates as a protective antioxidant and can be oxidized to hypoiodite, a potent oxidant involved in the host defense against microorganisms. This was possibly the original role of I in biological systems, before the appearance of TH in evolution. I per se participates in its own regulation, and new evidence indicates that it may be antineoplastic, anti-proliferative and cytotoxic in human cancer. Alterations in the expression of I transporters are associated with tumor development in a cancer-type-dependent manner and, accordingly, NIS, CFTR and ANO1 have been proposed as tumor markers. Radioactive iodide has been the mainstay adjuvant treatment for thyroid cancer for the last seven decades by virtue of its active transport by NIS. The rapid advancement of techniques that detect radioisotopes, in particular I, has made NIS a preferred target-specific theranostic agent.
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http://dx.doi.org/10.1530/ERC-17-0515DOI Listing
April 2018

MicroRNA-146b promotes PI3K/AKT pathway hyperactivation and thyroid cancer progression by targeting PTEN.

Oncogene 2018 06 22;37(25):3369-3383. Epub 2018 Jan 22.

Instituto de Investigaciones Biomédicas "Alberto Sols"; Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid (UAM), Madrid, Spain.

Recent studies have shown that miR-146b is the most upregulated microRNA in thyroid cancer and has a central role in cancer progression through mechanisms that remain largely unidentified. As phosphoinositide 3-kinase/protein kinase-B (PI3K/AKT) signaling is a fundamental oncogenic driver in many thyroid cancers, we explored a potential role for miR-146b and its target genes in PI3K/AKT activation. Among the predicted target genes of miR-146b, we found the tumor-suppressor phosphatase and tensin homolog (PTEN). Constitutive overexpression of miR-146b in thyroid epithelial cell lines significantly decreased PTEN mRNA and protein levels by direct binding to its 3'-UTR. This was accompanied by PI3K/AKT hyperactivation, leading to the exclusion of FOXO1 and p27 from the nucleus and a corresponding increase in cellular proliferation. Moreover, miR-146b overexpression led to protection from apoptosis and an increased migration and invasion potential, regulating genes involved in epithelial-mesenchymal transition. Notably, with the single exception of E-cadherin expression, all of these outcomes could be reversed by PTEN coexpression. Further analysis showed that miR-146b directly inhibits E-cadherin expression through binding to its 3'-UTR. Interestingly, miR-146b inhibition in human thyroid tumor xenografts, using a synthetic and clinically amenable molecule, blocked tumor growth when delivered intratumorally. Importantly, this inhibition increased PTEN protein levels. In conclusion, our data define a novel mechanism of PI3K/AKT hyperactivation and outline a regulatory role for miR-146b in suppressing PTEN expression, a frequent observation in thyroid cancer. Both events are related to a more aggressive tumoral phenotype. Targeting miR-146b therefore represents a promising therapeutic strategy for the treatment of this disease.
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http://dx.doi.org/10.1038/s41388-017-0088-9DOI Listing
June 2018

TAZ/WWTR1 Mediates the Pulmonary Effects of NKX2-1 Mutations in Brain-Lung-Thyroid Syndrome.

J Clin Endocrinol Metab 2018 03;103(3):839-852

Thyroid Molecular Laboratory, Institute for Medical and Molecular Genetics, La Paz University Hospital, Madrid, Spain.

Context: Identification of a frameshift heterozygous mutation in the transcription factor NKX2-1 in a patient with brain-lung-thyroid syndrome (BLTS) and life-threatening lung emphysema.

Objective: To study the genetic defect that causes this complex phenotype and dissect the molecular mechanism underlying this syndrome through functional analysis.

Methods: Mutational study by DNA sequencing, generation of expression vectors, site-directed mutagenesis, protein-DNA-binding assays, luciferase reporter gene assays, confocal microscopy, coimmunoprecipitation, and bioinformatics analysis.

Results: We identified a mutation [p.(Val75Glyfs*334)] in the amino-terminal domain of the NKX2-1 gene, which was functionally compared with a previously identified mutation [p.(Ala276Argfs*75)] in the carboxy-terminal domain in other patients with BLTS but without signs of respiratory distress. Both mutations showed similar protein expression profiles, subcellular localization, and deleterious effects on thyroid-, brain-, and lung-specific promoter activity. Coexpression of the coactivator TAZ/WWTR1 (transcriptional coactivator with PDZ-binding motif/WW domain-containing transcription regulator protein 1) restored the transactivation properties of p.(Ala276Argfs*75) but not p.(Val75Glyfs*334) NKX2-1 on a lung-specific promoter, although both NKX2-1 mutants could interact equally with TAZ/WWTR1. The retention of residual transcriptional activity in the carboxy-terminal mutant, which was absent in the amino-terminal mutant, allowed the functional rescue by TAZ/WWTR1.

Conclusions: Our results support a mechanistic model involving TAZ/WWTR1 in the development of human congenital emphysema, suggesting that this protein could be a transcriptional modifier of the lung phenotype in BLTS.
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http://dx.doi.org/10.1210/jc.2017-01241DOI Listing
March 2018

Patient Age-Associated Mortality Risk Is Differentiated by BRAF V600E Status in Papillary Thyroid Cancer.

J Clin Oncol 2018 02 14;36(5):438-445. Epub 2017 Dec 14.

Xiaopei Shen, Guangwu Zhu, Rengyun Liu, and Mingzhao Xing, Johns Hopkins University School of Medicine, Baltimore, MD; David Viola and Rossella Elisei, University of Pisa, Pisa; Efisio Puxeddu, University of Perugia, Perugia; Laura Fugazzola and Carla Colombo, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Istituto Auxologico Italiano and University of Milan, Milan; Caterina Mian, University of Padua; Federica Vianello, Veneto Institute of Oncology, IRCCS, Padua, Italy; Barbara Jarzab and Agnieszka Czarniecka, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland; Alfred K. Lam, Menzies Health Institute Queensland, Griffith University, Gold Coast, Queensland; Christine J. O'Neill, Mark S. Sywak, and Roderick Clifton-Bligh, The University of Sydney, Sydney, New South Wales, Australia; Linwah Yip, University of Pittsburgh School of Medicine, Pittsburgh, PA; Garcilaso Riesco-Eizaguirre, Hospital Universitario La Paz and Hospital Universitario de Móstoles; Garcilaso Riesco-Eizaguirre and Pilar Santisteban, Biomedical Research Institute "Alberto Sols," Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid; Garcilaso Riesco-Eizaguirre and Pilar Santisteban, Ciberonc, Health Institute Carlos III, Madrid, Spain; and Bela Bendlova and Vlasta Sýkorová, Institute of Endocrinology, Prague, Czech Republic.

Purpose For the past 65 years, patient age at diagnosis has been widely used as a major mortality risk factor in the risk stratification of papillary thyroid cancer (PTC), but whether this is generally applicable, particularly in patients with different BRAF genetic backgrounds, is unclear. The current study was designed to test whether patient age at diagnosis is a major mortality risk factor. Patients and Methods We conducted a comparative study of the relationship between patient age at diagnosis and PTC-specific mortality with respect to BRAF status in 2,638 patients (623 men and 2,015 women) with a median age of 46 years (interquartile range, 35 to 58 years) at diagnosis and a median follow-up time of 58 months (interquartile range, 26 to 107 months). Eleven medical centers from six countries participated in this study. Results There was a linear association between patient age and mortality in patients with BRAF V600E mutation, but not in patients with wild-type BRAF, in whom the mortality rate remained low and flat with increasing age. Kaplan-Meier survival curves rapidly declined with increasing age in patients with BRAF V600E mutation but did not decline in patients with wild-type BRAF, even beyond age 75 years. The association between mortality and age in patients with BRAF V600E was independent of clinicopathologic risk factors. Similar results were observed when only patients with the conventional variant of PTC were analyzed. Conclusion The long-observed age-associated mortality risk in PTC is dependent on BRAF status; age is a strong, continuous, and independent mortality risk factor in patients with BRAF V600E mutation but not in patients with wild-type BRAF. These results question the conventional general use of patient age as a high-risk factor in PTC and call for differentiation between patients with BRAF V600E and wild-type BRAF when applying age to risk stratification and management of PTC.
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http://dx.doi.org/10.1200/JCO.2017.74.5497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807010PMC
February 2018

BRAF V600E Mutation-Assisted Risk Stratification of Solitary Intrathyroidal Papillary Thyroid Cancer for Precision Treatment.

J Natl Cancer Inst 2018 04;110(4):362-370

Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD.

Background: Precise risk stratification-based treatment of solitary intrathyroidal papillary thyroid cancer (SI-PTC) that is larger than 1.0 cm and 4.0 cm or less is undefined.

Methods: A genetic-clinical risk study was performed on BRAF V600E in 955 patients (768 women and 187 men) with SI-PTC, with median age of 46 years and median clinical follow-up time of 64 months at 11 medical centers in six countries. The chi-square test or, for analyses with small numbers, Fisher's exact test was performed to compare recurrence rates. Recurrence-free probability was estimated by Kaplan-Meier (KM) analysis, and the independent effect of BRAF mutation on the recurrence was analyzed by Cox regression and Cox proportional hazard analyses. All statistical tests were two-sided.

Results: Recurrence of SI-PTC larger than 1.0 cm and 4.0 cm or less was 9.5% (21/221) vs 3.4% (11/319) in BRAF mutation vs wild-type BRAF patients, with a hazard ratio (HR) of 3.03 (95% confidence interval [CI] = 1.46 to 6.30) and a patient age- and sex-adjusted hazard ratio of 3.10 (95% CI = 1.49 to 6.45, P = .002). Recurrence rates of SI-PTC larger than 2.0 cm and 4.0 cm or less were 16.5% (13/79) vs 3.6% (5/139) in mutation vs wild-type patients (HR = 5.44, 95% CI = 1.93 to 15.34; and adjusted HR = 5.58, 95% CI = 1.96 to 15.85, P = .001). Recurrence rates of SI-PTC larger than 3.0 cm and 4 cm or less were 30.0% (6/20) vs 1.9% (1/54) in mutation vs wild-type patients (HR = 18.40, 95% CI = 2.21 to 152.98; and adjusted HR = 14.73, 95% CI = 1.74 to 124.80, P = .01). Recurrences of mutation-positive SI-PTC were comparable with those of counterpart invasive solitary PTC, around 20% to 30%, in tumors larger than 2.0 cm to 3.0 cm. BRAF mutation was associated with a statistically significant decrease in recurrence-free patient survival on KM analysis, particularly in SI-PTC larger than 2.0 cm and 4.0 cm or less. Similar results were obtained in conventional SI-PTC. The negative predictive values of BRAF mutation for recurrence were 97.8% (95% CI = 96.3% to 98.8%) for general SI-PTC and 98.2% (95% CI = 96.3% to 99.3%) for conventional SI-PTC.

Conclusions: BRAF V600E identifies a subgroup of SI-PTC larger than 1.0 cm and 4.0 cm or less, particularly tumors larger than 2.0 cm and 4.0 cm or less, that has high risk for recurrence comparable with that of invasive solitary PTC, making more aggressive treatment reasonable.
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http://dx.doi.org/10.1093/jnci/djx227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658860PMC
April 2018

Industry-Sponsored Satellite Symposia.

Eur Thyroid J 2017 Aug 12;6(Suppl 1):119-127. Epub 2017 Aug 12.

Madrid, Spain.

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http://dx.doi.org/10.1159/000477988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5611791PMC
August 2017

Key signaling pathways in thyroid cancer.

J Endocrinol 2017 Nov;235(2):R43-R61

Instituto de Investigaciones Biomédicas 'Alberto Sols'Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid (UAM), Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Instituto de Salud Carlos III (ISCIII), Madrid, Spain

Whole genome sequencing approaches have provided unprecedented insights into the genetic lesions responsible for the onset, progression and dedifferentiation of various types of thyroid carcinomas. Through these efforts, the MAPK and PI3K signaling cascades have emerged as the main activation pathways implicated in thyroid tumorigenesis. The nature of these essential pathways is highly complex, with hundreds of components, multiple points of crosstalk, different subcellular localizations and with the ability to potentially regulate many cellular processes. Small-molecule inhibitors targeting key kinases of these pathways hold great promise as novel therapeutics and several have reached clinical trials. However, while some remarkable responses have been reported, the development of resistance remains a matter of concern and limits the benefit for patients. In this review, we discuss the latest findings on the major components of the MAPK and PI3K pathways, including their mechanisms of activation in physiological and pathological contexts, their genetic alterations with respect to the different types of thyroid carcinomas and the more relevant drugs designed to block their activity.
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http://dx.doi.org/10.1530/JOE-17-0266DOI Listing
November 2017

The Prognostic Value of Tumor Multifocality in Clinical Outcomes of Papillary Thyroid Cancer.

J Clin Endocrinol Metab 2017 09;102(9):3241-3250

Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology, Diabetes & Metabolism, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287.

Context: Multifocality is often treated as a risk factor for papillary thyroid cancer (PTC), prompting aggressive treatments, but its prognostic value remains unestablished.

Objective: To investigate the role of tumor multifocality in clinical outcomes of PTC.

Methods: Multicenter study of the relationship between multifocality and clinical outcomes of PTC in 2638 patients (623 men and 2015 women) with median [interquartile range (IQR)] age of 46 (35 to 58) years and median (IQR) follow-up time of 58 (26 to 107) months at 11 medical centers in six countries. Surveillance, Epidemiology and End Results (SEER) data were used for validation.

Results: Disease recurrence in multifocal and unifocal PTC was 198 of 1000 (19.8%) and 221 of 1624 (13.6%) (P < 0.001), with a hazard ratio of 1.55 [95% confidence interval (CI), 1.28 to 1.88], which became insignificant at 1.13 (95% CI, 0.93 to 1.37) on multivariate adjustment. Similar results were obtained in PTC variants: conventional PTC, follicular-variant PTC, tall-cell PTC, and papillary thyroid microcarcinoma. There was no association between multifocality and mortality in any of these PTC settings, whereas there was a strong association between classic risk factors and cancer recurrence or mortality, which remained significant after multivariate adjustment. In 1423 patients with intrathyroidal PTC, disease recurrence was 20 of 455 (4.4%) and 41 of 967 (4.2%) (P = 0.892) and mortality was 0 of 455 (0.0%) and 3 of 967 (0.3%) (P = 0.556) in multifocal and unifocal PTC, respectively. The results were reproduced in 89,680 patients with PTC in the SEER database.

Conclusions: Tumor multifocality has no independent risk prognostic value in clinical outcomes of PTC; its indiscriminate use as an independent risk factor, prompting overtreatments of patients, should be avoided.
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http://dx.doi.org/10.1210/jc.2017-00277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587077PMC
September 2017

Pax8 controls thyroid follicular polarity through cadherin-16.

J Cell Sci 2017 01 25;130(1):219-231. Epub 2016 Oct 25.

Department of Endocrine and Nervous System Physiopathology, Instituto de Investigaciones Biomédicas 'Alberto Sols', Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid (CSIC-UAM), Madrid 28029, Spain

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 β1-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.
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http://dx.doi.org/10.1242/jcs.184291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5394772PMC
January 2017

ENDOCRINE TUMOURS: Advances in the molecular pathogenesis of thyroid cancer: lessons from the cancer genome.

Eur J Endocrinol 2016 Nov;175(5):R203-17

Instituto de Investigaciones Biomédicas "Alberto Sols" Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid (CSIC-UAM)Madrid, Spain

Thyroid cancer is the most common endocrine malignancy giving rise to one of the most indolent solid cancers, but also one of the most lethal. In recent years, systematic studies of the cancer genome, most importantly those derived from The Cancer Genome Altas (TCGA), have catalogued aberrations in the DNA, chromatin, and RNA of the genomes of thousands of tumors relative to matched normal cellular genomes and have analyzed their epigenetic and protein consequences. Cancer genomics is therefore providing new information on cancer development and behavior, as well as new insights into genetic alterations and molecular pathways. From this genomic perspective, we will review the main advances concerning some essential aspects of the molecular pathogenesis of thyroid cancer such as mutational mechanisms, new cancer genes implicated in tumor initiation and progression, the role of non-coding RNA, and the advent of new susceptibility genes in thyroid cancer predisposition. This look across these genomic and cellular alterations results in the reshaping of the multistep development of thyroid tumors and offers new tools and opportunities for further research and clinical development of novel treatment strategies.
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http://dx.doi.org/10.1530/EJE-16-0202DOI Listing
November 2016

β-catenin signaling is required for RAS-driven thyroid cancer through PI3K activation.

Oncotarget 2016 Aug;7(31):49435-49449

Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas (CSIC) y Universidad Autónoma de Madrid (UAM), Madrid, Spain.

Mutations in β-catenin are traditionally described as late events in thyroid cancer progression. However, the functional implications of β-catenin dysregulation in the context of tumor initiating events remain unclear. The aim of this work was to investigate whether the two main oncogenic drivers in thyroid cancer, RAS and BRAF, could activate the Wnt/β-catenin pathway. Expression of HRASV12 but not BRAFV600E in thyroid cells induced β-catenin nuclear localization, increased β-catenin-dependent transcriptional activity and inhibited GSK3β. In a panel of human thyroid cancer cell lines representative of the main genetic events in thyroid cancer, β-catenin activation was highly dependent on PI3K/AKT activity through its phosphorylation at S552, but not on MAPK. Silencing of β-catenin expression in cell lines led to a dramatic reduction in proliferation due to an induction of senescence, which was concordant with a reduction in tumor size in nude mice. Moreover, β-catenin silencing suppressed the expression of EMT-related genes and reduced the invasive capacity of the tumor cells. In conclusion, this work demonstrates that RAS-driven tumors induce PI3K/AKT-dependent β-catenin activation.
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http://dx.doi.org/10.18632/oncotarget.10356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226519PMC
August 2016