Expression Of Thyroid Differentiation Markers Research Articles

Targeting miR-31 represses tumourigenesis and dedifferentiation of BRAFV600E-associated thyroid carcinoma.

BRAFV600E is the most common genetic mutation in differentiated thyroid cancer (DTC) occurring in 60% of patients and drives malignant tumour cell phenotypes including proliferation, metastasis and immune-escape. BRAFV600E-mutated papillary thyroid cancer (PTC) also displays greatly reduced expression of thyroid differentiation markers, thus tendency to radioactive iodine (RAI) refractory and poor prognosis. Therefore, understanding the molecular mechanisms and main oncogenic events underlying BRAFV600E will guide future therapy development. Bioinformatics and clinical specimen analyses, genetic manipulation of BRAFV600E-induced PTC model, functional and mechanism exploration guided with transcriptomic screening, as well as systematic rescue experiments were applied to investigate miR-31 function within BRAFV600E-induced thyroid cancer development. Besides, nanoparticles carrying miR-31 antagomirs were testified to alleviate 131I iodide therapy on PTC models. We identify miR-31 as a significantly increased onco-miR in BRAFV600E-associated PTC that promotes tumour progression, metastasis and RAI refractoriness via sustained Wnt/β-catenin signalling. Mechanistically, highly activated BRAF/MAPK pathway induces miR-31 expression via c-Jun-mediated transcriptional regulation across in vitro and transgenic mouse models. MiR-31 in turn facilitates β-catenin stabilisation via directly repressing tumour suppressors CEBPA and DACH1, which direct the expression of multiple essential Wnt/β-catenin pathway inhibitors. Genetic functional assays showed that thyroid-specific knockout of miR-31 inhibited BRAFV600E-induced PTC progression, and strikingly, enhanced expression of sodium-iodide symporter and other thyroid differentiation markers, thus promoted 131I uptake. Nanoparticle-mediated application of anti-miR-31 antagomirs markedly elevated radio-sensitivity of BRAFV600E-induced PTC tumours to 131I therapy, and efficiently suppressed tumour progression in the pre-clinical mouse model. Our findings elucidate a novel BRAF/MAPK-miR-31-Wnt/β-catenin regulatory mechanism underlying clinically BRAFV600E-associated DTC tumourigenesis and dedifferentiation, also highlight a potential adjuvant therapeutic strategy for advanced DTC.

Abstract 1219: Establishment and characterization of amitrole-induced mouse thyroid adenoma-derived cell lines

Abstract Some types of thyroid cancers are known to arise from thyroid adenomas, as a malignant transition from benign adenoma, as proposed in the multi-step carcinogenesis model. Thyroid cancer studies using human thyroid cancer cell lines have been intensely performed by many researchers. However, mouse thyroid adenoma cell lines have not been well established and characterized. In order to obtain further insights into thyroid carcinogenesis process, we established five cell lines from thyroid adenomas developed in mice treated with goitrogen amitrole. Among them, two cell lines, CAT458/458s (CAT: cells from amitrole treated thyroids; CAT458s: a sub-line from CAT458) and CAT459 were found to be positive for E-cadherin and Claudin-1, representative epithelial markers, and negative for Vimentin, a mesenchymal marker, suggesting that they originated from thyroid epithelial cells. We found higher expression of DUOX1, a member of NADPH oxidase involved in thyroid hormone synthesis, in both CAT458s and 459 cells than normal mouse thyroids, presumably due to an unknown mechanism caused by amitrole treatment. NKX2-1, a master regulator of thyroid development, is not expressed in CAT458s cells while NKX2-1 is abundantly expressed in CAT459 cells. Consistent with this finding, NKX2-1-positive CAT459 cells showed higher mRNA expression of thyroid differentiation markers including Tg, Duox1, and Duox2. NKX2-1 overexpression in NKX2-1-negative CAT458s cells using a lentiviral infection system induced mRNA expressions of these thyroid differentiation marker genes, suggesting that partial thyroid differentiation of the adenoma cells is induced by NKX2-1. TSH and IGF-I are known to work synergistically for thyroid growth. However, Tshr mRNA expression is not detected in either CAT458s or 459 cells, suggesting that TSH signal is not responsible for their growth. On the other hand, IGF-IR protein is expressed and IGF-I induces AKT and S6 phosphorylation in both cells. We further tried to assess somatic gene mutations involved in adenoma development by whole-genome sequencing (WGS). WGS analysis revealed that somatic missense mutations are not found in genes known to be involved in thyroid carcinogenesis and cancer progression, such as Braf, Trp53, Ret, Tert, Hras, Kras, and Nras. This finding suggests that prominent oncogenic mutations are not required for thyroid adenoma development that may later progress to carcinoma. Currently we are validating several missense mutations, including mutations in tumor suppressor genes Atm and Tgfb1, found in these cell lines. In conclusion, we established mouse thyroid adenoma cell lines with different differentiation and molecular profiles. CAT458s and 459 cells would provide a tool to further clarify the process of thyroid adenoma development and thyroid differentiation. Citation Format: Yo-Taro Shirai, Nobuo Hoshi, Huaitian Liu, Maxwell P. Lee, Shioko Kimura. Establishment and characterization of amitrole-induced mouse thyroid adenoma-derived cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1219.

Thyroid Hormone Receptor Beta Induces a Tumor-Suppressive Program in Anaplastic Thyroid Cancer.

The thyroid hormone receptor beta (TRβ), a key regulator of cellular growth and differentiation, is frequently dysregulated in cancers. Diminished expression of TRβ is noted in thyroid, breast, and other solid tumors and is correlated with more aggressive disease. Restoration of TRβ levels decreased tumor growth supporting the concept that TRβ could function as a tumor suppressor. Yet, the TRβ tumor suppression transcriptome is not well delineated and the impact of TRβ is unknown in aggressive anaplastic thyroid cancer (ATC). Here, we establish that restoration of TRβ expression in the human ATC cell line SW1736 (SW-TRβ) reduces the aggressive phenotype, decreases cancer stem cell populations and induces cell death in a T3-dependent manner. Transcriptomic analysis of SW-TRβ cells via RNA sequencing revealed distinctive expression patterns induced by ligand-bound TRβ and revealed novel molecular signaling pathways. Of note, liganded TRβ repressed multiple nodes in the PI3K/AKT pathway, induced expression of thyroid differentiation markers, and promoted proapoptotic pathways. Our results further revealed the JAK1-STAT1 pathway as a novel, T3-mediated, antitumorigenic pathway that can be activated in additional ATC lines. These findings elucidate a TRβ-driven tumor suppression transcriptomic signature, highlight unexplored therapeutic options for ATC, and support TRβ activation as a promising therapeutic option in cancers. IMPLICATIONS: TRβ-T3 induced a less aggressive phenotype and tumor suppression program in anaplastic thyroid cancer cells revealing new potential therapeutic targets.

The Transcription Factor NF-κB Mediates Thyrotropin-Stimulated Expression of Thyroid Differentiation Markers.

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.

A ceRNA Circuitry Involving the Long Noncoding RNA Klhl14-AS, Pax8, and Bcl2 Drives Thyroid Carcinogenesis.

Klhl14-AS is a long noncoding RNA expressed since early specification of thyroid bud and is the most enriched gene in the mouse thyroid primordium at E10.5. Here, we studied its involvement in thyroid carcinogenesis by analyzing its expression in cancer tissues and different models of neoplastic transformation. Compared with normal thyroid tissue and cells, Klhl14-AS was significantly downregulated in human thyroid carcinoma tissue specimens, particularly the anaplastic histotype, thyroid cancer cell lines, and rodent models of thyroid cancer. Downregulating the expression of Klhl14-AS in normal thyroid cells decreased the expression of thyroid differentiation markers and cell death and increased cell viability. These effects were mediated by the binding of Klhl14-AS to two miRNAs, Mir182-5p and Mir20a-5p, which silenced Pax8 and Bcl2, both essential players of thyroid differentiation. MIR182-5p and MIR20a-5p were upregulated in human thyroid cancer and thyroid cancer experimental models and their effects on Pax8 and Bcl2 were rescued by Klhl14-AS overexpression, confirming Klhl14-AS as a ceRNA for both Pax8 and Bcl2. This work connects deregulation of differentiation with increased proliferation and survival in thyroid neoplastic cells and highlights a novel ceRNA circuitry involving key regulators of thyroid physiology. SIGNIFICANCE: This study describes a new ceRNA with potential tumor suppression activity and helps us better understand the regulatory mechanisms during thyroid differentiation and carcinogenesis.

Double knock-out of Hmga1 and Hipk2 genes causes perinatal death associated to respiratory distress and thyroid abnormalities in mice

The serine–threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) modulates important cellular functions during development, acting as a signal integrator of a wide variety of stress signals, and as a regulator of transcription factors and cofactors. We have previously demonstrated that HIPK2 binds and phosphorylates High-Mobility Group A1 (HMGA1), an architectural chromatinic protein ubiquitously expressed in embryonic tissues, decreasing its binding affinity to DNA. To better define the functional role of HIPK2 and HMGA1 interaction in vivo, we generated mice in which both genes are disrupted. About 50% of these Hmga1/Hipk2 double knock-out (DKO) mice die within 12 h of life (P1) for respiratory failure. The DKO mice present an altered lung morphology, likely owing to a drastic reduction in the expression of surfactant proteins, that are required for lung development. Consistently, we report that both HMGA1 and HIPK2 proteins positively regulate the transcriptional activity of the genes encoding the surfactant proteins. Moreover, these mice display an altered expression of thyroid differentiation markers, reasonably because of a drastic reduction in the expression of the thyroid-specific transcription factors PAX8 and FOXE1, which we demonstrate here to be positively regulated by HMGA1 and HIPK2. Therefore, these data indicate a critical role of HIPK2/HMGA1 cooperation in lung and thyroid development and function, suggesting the potential involvement of their impairment in the pathogenesis of human lung and thyroid diseases.

Aging Is Associated with Low Thyroid State and Organ-Specific Sensitivity to Thyroxine

Background: Serum thyroid state in older adults correlates with extended longevity. We hypothesized that age impacts not only systemic but also organ-specific thyroid state and response to thyroxine (T4). Methods: Young (3 months) and old (23 months) male mice were analyzed at baseline and after acute T4 challenge. Age effects on circulating thyrotropin (TSH) and thyroid hormone (TH) concentrations, transcript expression in the pituitary and thyroid were compared with organ-specific responses characterized by hepatic and cardiac content of TH and TH metabolites and expression of TH-target genes, as well as hepatic deiodinase 1 activity. Results: Circulating TH concentrations and hepatic and cardiac TH content were lower in old versus young mice. After injection with T4, conversion of T4 to triiodothyronine was decreased in old mice while TH transport in liver and heart was not affected. Organ-specific TH response was augmented in old mice in liver but not heart, indicating age- and tissue-specific sensitivity to TH. A compensatory increase of thyroid stimulating hormone subunit beta expression in the pituitary and increased serum TSH concentrations, but reduced expression of thyroid differentiation markers were found in old mice. Conclusions: We suggest that a reduced activity of the aged thyroid is responsible for the systemic low TH state in old mice. Further, divergent TH metabolism and tissue response in liver and heart occur after T4 treatment in an aged organism. These rodent data are in agreement with a much narrower window for T4 substitution in the older adults to avoid overtreatment.

Ethacridine Induces Apoptosis and Differentiation in Thyroid Cancer Cells In Vitro.

Ethacridine is used as a topical antiseptic as well as for second-trimester abortion. Recent studies showed that ethacridine is an inhibitor of poly(ADP-ribose) glycohydrolase (PARG) and an activator of the transcriptional coactivator with PDZ-binding motif (TAZ). This study examined the effects of ethacridine on thyroid cancer cells. Thyroid cancer cell lines (FTC133 and SW1736) and thyroid follicular epithelial cells (Nthy-ori 3-1) were treated with ethacridine. Viability, clonogenicity, cell-cycle distribution, and apoptosis were evaluated. The expression of thyroid differentiation markers (TTF-1, PAX8, and NIS) was determined by real-time PCR. Ethacridine suppressed cell growth and clonogenic ability of thyroid cancer cells in a time- and dose-dependent manner (p<0.001). No cell-cycle arrest was found, but ethacridine dose-dependently induced apoptosis of thyroid cancer cells (p<0.001). The PAX8 and NIS expressions were significantly increased in SW1736 (3.41-fold and 1.53-fold, respectively) and Nthy-ori 3-1 cells (2.73-fold and 4.12-fold, respectively). Ethacridine elicits apoptotic cell death in thyroid cancer cells and promotes differentiation in a subset of thyroid follicular cells.

NKX2.5 is expressed in papillary thyroid carcinomas and regulates differentiation in thyroid cells

BackgroundNKX2.5 is a transcription factor transiently expressed during thyroid organogenesis. Recently, several works have pointed out the oncogenic role of NKX2.5 in a variety of tumors. We therefore hypothesized that NKX2.5 could also play a role in thyroid cancer.MethodsThe validation of NKX2.5 expression was assessed by immunohistochemistry analysis in a Brazilian case series of 10 papillary thyroid carcinoma (PTC) patients. Then, the long-term prognostic value of NKX2.5 and its correlation with clinicopathologic features of 51 PTC patients was evaluated in a cohort with 10-years follow-up (1990–1999). Besides, the effect of NKX2.5 overexpression on thyroid differentiation markers and function was also investigated in a non-tumor thyroid cell line (PCCL3).ResultsNKX2.5 was shown to be expressed in most PTC samples (8/10, case series; 27/51, cohort). Patients who had tumors expressing NKX2.5 showed lower rates of persistence/recurrence (p = 0.013). Overexpression of NKX2.5 in PCCL3 cells led to: 1) downregulation of thyroid differentiation markers (thyrotropin receptor, thyroperoxidase and sodium-iodide symporter); 2) reduced iodide uptake; 3) increased extracellular H2O2 generation, dual oxidase 1 mRNA levels and activity of DuOx1 promoter.ConclusionsIn summary, NKX2.5 is expressed in most PTC samples analyzed and its presence correlates to better prognosis of PTC. In vitro, NKX2.5 overexpression reduces the expression of thyroid differentiation markers and increases ROS production. Thus, our data suggests that NKX2.5 could play a role in thyroid carcinogenesis.

Distinctive Desmoplastic 3D Morphology Associated With BRAFV600E in Papillary Thyroid Cancers.

Although 60% of papillary thyroid carcinomas are BRAFV600E mutant (PTCV600E), the increased aggressiveness of these cancers is still debated. For PTCV600E we aimed to further characterize the extent of the stroma and its activation, the three-dimensional (3D) tumor-stroma interface, and the proliferation rates of tumor and stromal fibroblasts. We analyzed exomes, transcriptomes, and images of 364 papillary thyroid carcinoma (PTCs) from The Cancer Genome Atlas (TCGA), including 211 PTCV600E; stained 22 independent PTCs for BRAFV600E and Ki67; sequenced the exomes and stained BRAFV600E in 5 primary tumor blocks and 4 nodal metastases from one patient with PTCV600E; and reconstructed the 3D volumes of one tumor and one metastatic block at histological resolution. In TCGA, BRAFV600E was associated with higher expression of proliferation markers and lower expression of thyroid differentiation markers, independently of tumor purity. Moreover, PTCV600E, in line with their overall lower purity, also had higher expression of fibroblast- and T cell-associated genes and presented more fibrosis. Tumor cells that appeared disconnected on two-dimensional histological slices were revealed to be part of a unique tumor component in the 3D reconstructed microvolumes, and they formed a surprisingly complex connected space, infiltrating a proliferative stroma. Finally, in our PTC set, both stromal fibroblasts and tumor cells presented higher proliferation rates in PTCV600E. Our results support the increased aggressiveness associated with BRAFV600E in PTC and shed light on the important role of the stroma in tumor expansion. The greater and more active fibrotic component predicts better efficiency of combined targeted treatments, as previously proposed for melanomaV600E.

Maternal Exposure to Iodine Excess Throughout Pregnancy and Lactation Induces Hypothyroidism in Adult Male Rat Offspring

This study aimed to investigate the consequences of maternal exposure to iodine excess (IE; 0.6 mg NaI/L) throughout pregnancy and lactation on the hypothalamus-pituitary-thyroid axis of the male offspring in adulthood. Maternal IE exposure increased hypothalamic Trh mRNA expression and pituitary Tsh expression and secretion in the adult male offspring. Moreover, the IE-exposed offspring rats presented reduced thyroid hormones levels, morphological alterations in the thyroid follicles, increased thyroid oxidative stress and decreased expression of thyroid differentiation markers (Tshr, Nis, Tg, Tpo, Mct8) and thyroid transcription factors (Nkx2.1, Pax8). Finally, the data presented here strongly suggest that epigenetic mechanisms, as increased DNA methylation, augmented DNA methyltransferases expression, hypermethylation of histone H3, hypoaceylation of histones H3 and H4, increased expression/activity of histone deacetylases and decreased expression/activity of histone acetyltransferases are involved in the repression of thyroid gene expression in the adult male offspring. In conclusion, our results indicate that rat dams’ exposure to IE during pregnancy and lactation induces primary hypothyroidism and triggers several epigenetic changes in the thyroid gland of their male offspring in adulthood.

IFN-α Mediates the Development of Autoimmunity both by Direct Tissue Toxicity and through Immune Cell Recruitment Mechanisms

IFN-α is known to play a key role in autoimmunity, but the mechanisms are uncertain. Although the induction of autoimmunity by IFN-α is consistent with primarily immunomodulatory effects, the high frequency of nonautoimmune inflammation suggests other mechanisms. We used thyroiditis as a model to dissect these possibilities. IFN-α treatment of cultured thyrocytes increased expression of thyroid differentiation markers, thyroglobulin, thyroid-stimulating hormone receptor, thyroid peroxidase, and sodium iodide transporter. RNAseq analysis demonstrated that pathways of Ag presentation, pattern recognition receptors, and cytokines/chemokines were also stimulated. These changes were associated with markedly increased nonapoptotic thyroid cell death, suggesting direct toxicity. To corroborate these in vitro findings, we created transgenic mice with thyroid-specific overexpression of IFN-α under control of the thyroglobulin promoter. Transgenic mice developed marked inflammatory thyroid destruction associated with immune cell infiltration of thyroid and surrounding tissues leading to profound hypothyroidism, findings consistent with our in vitro results. In addition, transgenic mice thyroids showed upregulation of pathways similar to those observed in cultured thyrocytes. In particular, expression of granzyme B, CXCL10, a subset of the tripartite motif-containing family, and other genes involved in recruitment of bystander cytotoxic immune responses were increased. Pathways associated with apoptosis and autophagy were not induced. Taken together, our data demonstrate that the induction of tissue inflammation and autoimmunity by IFN-α involves direct tissue toxic effects as well as provocation of destructive bystander immune responses.

Modulation of retrovirally driven therapeutic genes by mutant TP53 in anaplastic thyroid carcinoma

We previously demonstrated that restoration of TP53 activity in anaplastic thyroid carcinoma inhibits cell growth and induces expression of thyroid differentiation markers. Here, we investigated whether TP53 status may condition the expression of therapeutic genes driven by retroviral LTR or tissue-specific enhancer elements. The TP53-defective ARO anaplastic thyroid carcinoma cells were transfected with TP53(Val135), which exhibits wild-type activity at 32 degrees C, and transduced with retroviral vectors, in which therapeutic genes were driven either by wild-type LTR or by a reshuffled LTR containing thyroglobulin (TG) enhancer. Both at 37 and 32 degrees C, expression of transgenes driven by TG enhancer was 10-fold lower than that obtained with wild-type LTR retroviral vector. TP53(Val135) transfer into ARO cells repressed transcription from wild-type LTR but increased expression of TG-driven therapeutic genes. This effect was markedly enhanced by cell culture at 32 degrees C and by TSH treatment. Cytotoxic effects shown after ganciclovir treatment paralleled therapeutic gene expression levels. In conclusion, TP53 status in the tumor cell can influence expression of therapeutic genes. When using retroviral-vector-based gene therapy, wild-type LTR vectors should be employed to target TP53-defective tumors, whereas thyroid-specific promoters should be used for transcriptional targeting of thyroid carcinomas carrying wild-type TP53.