Alterations in Body Weight and Composition in Patients With Graves' Disease Treated with Total Thyroidectomy.

OBJECTIVE: Thyrotoxicosis causes excess energy expenditure, resulting in weight loss, despite increased appetite, and changes in body composition, which are typically reversible with the normalization of thyroid hormone levels. However, patients with hyperthyroidism due to Graves' disease are sometimes hesitant to undergo treatment because of the perceived morbidity associated with weight gain. Therefore, obtaining data to explain the details of such weight gain to these patients is important. This study aimed to investigate changes in body weight and composition in patients with Graves' disease after total thyroidectomy. METHODS: In total, 21 patients with Graves' disease who underwent total thyroidectomy were enrolled. Among them, nine patients were hyperthyroid (group A) and 12 were euthyroid (group B, control) immediately before surgery. Body weight, height, and body composition using bioelectrical impedance were measured preoperatively and five months postoperatively. RESULTS: In all patients, body weight, body mass index, and skeletal muscle mass, but not fat mass, significantly increased postoperatively. In individual groups, a significant increase in skeletal muscle and fat masses was observed solely in groups A and B, respectively. Furthermore, a significant positive correlation between preoperative thyroid function and differences in skeletal muscle mass preoperatively and postoperatively was found. CONCLUSION: Our study shows that the normalization of thyroid function using thyroidectomy in patients with Graves' disease is accompanied by weight gain, mainly due to an increase in skeletal muscle mass. These data are clinically significant because they enable physicians to explain to patients that weight gain after surgical treatment for Graves' disease is favorable and reassure them of their concern.

Characterization of metabolic reprogramming by metabolomics in the oncocytic thyroid cancer cell line XTC.UC1.

Oncocytic thyroid cancer is characterized by the aberrant accumulation of abnormal mitochondria in the cytoplasm and a defect in oxidative phosphorylation. We performed metabolomics analysis to compare metabolic reprogramming among the oncocytic and non-oncocytic thyroid cancer cell lines XTC.UC1 and TPC1, respectively, and a normal thyroid cell line Nthy-ori 3-1. We found that although XTC.UC1 cells exhibit higher glucose uptake than TPC1 cells, the glycolytic intermediates are not only utilized to generate end-products of glycolysis, but also diverted to branching pathways such as lipid metabolism and the serine synthesis pathway. Glutamine is preferentially used to produce glutathione to reduce oxidative stress in XTC.UC1 cells, rather than to generate α-ketoglutarate for anaplerotic flux into the TCA cycle. Thus, growth, survival and redox homeostasis of XTC.UC1 cells rely more on both glucose and glutamine than do TPC1 cells. Furthermore, XTC.UC1 cells contained higher amounts of intracellular amino acids which is due to higher expression of the amino acid transporter ASCT2 and enhanced autophagy, thus providing the building blocks for macromolecules and energy production. These metabolic alterations are required for oncocytic cancer cells to compensate their defective mitochondrial function and to alleviate excess oxidative stress.

The Effect of Long-Term Inorganic Iodine on Intrathyroidal Iodothyronine Content and Gene Expression in Mice with Graves' Hyperthyroidism.

Background: The main molecular mechanism underlying acute suppression of iodine organification in normal thyroids after an excessive iodine load, that is, the Wolff-Chaikoff effect, is assumed to be suppression of iodine oxidation and iodothyronine synthesis. However, the mechanism underlying chronic antithyroid action of inorganic iodine in Graves' disease is not fully understood. Using a mouse model of Graves' hyperthyroidism, we examined changes in iodothyronine content and gene expression profiles in the thyroid glands after inorganic iodine loading. Materials and Methods: Graves' hyperthyroidism was induced and maintained in BALB/c mice by repeated immunizations of recombinant adenovirus expressing the human thyrotropin (TSH) receptor A-subunit. Hyperthyroid mice were left untreated (GD-C; n = 8) or treated with inorganic iodine for 12 weeks (GD-NaI; n = 8). We used unimmunized BALB/c mice as a control group (n = 10). In each mouse, serum thyroxine (T4) levels were measured with enzyme-linked immunosorbent assay (ELISA) at 4-week intervals. The intrathyroidal iodothyronine content and gene expression levels were, respectively, evaluated by mass spectrometry and RNA sequencing (RNA-seq) at the end of the experimental period. Results: Serum T4 levels in the GD-C group remained higher than in the control group, whereas those in the GD-NaI group declined to normal levels during the experimental period. Intrathyroidal triiodothyronine (T3), reverse T3 (rT3), and T4 contents in the GD-C group were higher than the control group, and rT3 and T4 were further increased in the GD-NaI group. The observed alterations in iodothyronine levels in the thyroid and sera may be explained by altered expression levels of genes for iodothyronine biosynthetic molecules, their transporter, and deiodinases. Conclusion: In this mouse model of hyperthyroidism, higher intrathyroidal accumulation of T4 and reduced gene expression data of iodothyronine transporters in the GD-NaI group suggest that chronic antithyroid action of iodine in Graves' disease involves suppression of hormone secretion.

Reprogramming of Cellular Metabolism and Its Therapeutic Applications in Thyroid Cancer.

Metabolism is a series of life-sustaining chemical reactions in organisms, providing energy required for cellular processes and building blocks for cellular constituents of proteins, lipids, carbohydrates and nucleic acids. Cancer cells frequently reprogram their metabolic behaviors to adapt their rapid proliferation and altered tumor microenvironments. Not only aerobic glycolysis (also termed the Warburg effect) but also altered mitochondrial metabolism, amino acid metabolism and lipid metabolism play important roles for cancer growth and aggressiveness. Thus, the mechanistic elucidation of these metabolic changes is invaluable for understanding the pathogenesis of cancers and developing novel metabolism-targeted therapies. In this review article, we first provide an overview of essential metabolic mechanisms, and then summarize the recent findings of metabolic reprogramming and the recent reports of metabolism-targeted therapies for thyroid cancer.

MIEAP and ATG5 are tumor suppressors in a mouse model of BRAFV600E-positive thyroid cancer.

Mitochondria-eating protein (MIEAP) is a molecule important for non-canonical mitophagy and thought to be a tumor suppressor. Our previous study found that MIEAP expression is defective in thyroid oncocytomas, irrespective of being benign or malignant, and also in non-oncocytic thyroid cancers. Thyroid oncocytomas are composed of large polygonal cells with eosinophilic cytoplasm that is rich in abnormal mitochondria. Thus, our data indicate that, together with increased mitochondrial biogenesis that compensates for the dysfunction of the mitochondria, MIEAP plays a critical role in the accumulation of mitochondria in thyroid oncocytic tumors, whereas a defective MIEAP expression alone is not sufficient for mitochondrial accumulation in non-oncocytic cancers with normal mitochondria. To clarify whether MIEAP is a tumor suppressor in the thyroids and whether MIEAP knockout (KO) alone is sufficient for the oncocytic phenotype and also to extend our effort toward canonical mitophagy (a selective autophagy), we here conducted mouse studies using genetically engineered mice. BrafCA/wt mice developed thyroid cancers 1 year after intrathyroidal injection of adenovirus expressing Cre, while cancer development was observed at 6 months in adenovirus-Cre-injected BrafCA/wt;MieapKO/KO and BrafCA/wt;Atg5flox/flox mice [where autophagy-related 5 (ATG5) is a component of autophagic machinery], although KO of either molecule alone was not sufficient for cancer development. These data demonstrate that MIEAP or ATG5 KO accelerated thyroid cancer development. However, cancers in adenovirus-Cre-injected BrafCA/wt ;MieapKO/KO and BrafCA/wt ;Atg5flox/flox mice were not oncocytic. In conclusion, we here show that MIEAP and ATG5 are both tumor suppressors in thyroid carcinogenesis, but as we have anticipated from our previous data, KO of either molecule does not confer the oncocytic phenotype to BRAFV600E-positive thyroid cancers. The combination of disruptive mitochondrial function and impaired mitochondrial quality control may be necessary to establish a mouse model of thyroid oncocytoma.

Thyrotropin receptor antagonists and inverse agonists, and their potential application to thyroid diseases.

The thyrotropin receptor (TSHR) plays critical roles in thyroid growth and function and in the pathogenesis of several thyroid diseases including Graves' hyperthyroidism and ophthalmopathy, non-autoimmune hyperthyroidism and thyroid cancer. Several low-molecular weight compounds (LMWCs) and anti-TSHR monoclonal antibodies (mAbs) with receptor antagonistic and inverse agonistic activities have been reported. The former binds to the pocket formed by the receptor transmembrane bundle, and the latter to the extracellular TSH binding site. Both are effective inhibitors of TSH/thyroid stimulating antibody-stimulated cAMP and/or hyaluronic acid production in TSHR-expressing cells. Anti-insulin-like growth factor 1 inhibitors are also found to inhibit TSHR signaling. Each agent has advantages and disadvantages; for example, mAbs have a higher affinity and longer half-life but are more costly than LMWCs. At present, mAbs appear most promising, yet the development of more efficacious LMWCs is desirable. These agents are anticipated to be efficacious not only for the above-mentioned diseases but also for resistance to thyroid hormone and have utility for thyroid cancer radionuclide scintigraphy/therapy as a new theranostic.

Re-evaluation of the role of autophagy in thyroid cancer treatment.

Numerous studies have examined the role of autophagy in thyroid cancer treatment; however there are discrepancies among the reported data, with some showing the pro-survival and others the anti-survival effects of autophagy. These discrepant results appear to be at least in part due to insufficient analyses or data misinterpretation as well as improper assessments of autophagic activity. Therefore, the present study re-evaluated the regulation of autophagic activity by various anticancer modalities and examined the role of autophagy in thyroid cancer treatment in three thyroid cancer cell lines (TPC1, ACT1 and KTC1). The immunofluorescence and DalGreen findings demonstrated that cisplatin, irradiation and sorafenib were all autophagy inducers as previously reported, but, unlike previous studies using thyroid cancer cells, doxorubicin acted as an inhibitor. KTC1 cells are unique because they only responded to cisplatin. The efficacy of anticancer therapeutics was significantly higher in chloroquine or 3-methyladenine-treated autophagy-defective cells than in autophagy-competent cells, thereby indicating the pro-survival effect of autophagy induced by anticancer therapeutics, which is partly due to inhibition of apoptosis. Thus, the present findings relating to several anticancer therapeutics and three thyroid cancer cell lines demonstrate the pro-survival effect of autophagy in thyroid cancer treatment. Although the present study only involved cell lines, it provides evidence for the beneficial combination of the anticancer therapeutic modalities with autophagy inhibitors, and proposes that autophagy inhibitors may serve as a possible adjunctive therapy for thyroid cancer.

Age-dependent effects on radiation-induced carcinogenesis in the rat thyroid.

Childhood radiation exposure is a known thyroid cancer risk factor. This study evaluated the effects of age on radiation-induced thyroid carcinogenesis in rats irradiated with 8 Gy X-rays. We analyzed cell proliferation, cell death, DNA damage response, and autophagy-related markers in 4-week-old (4W) and 7-month-old (7M) rats and the incidence of thyroid tumors in 4W, 4-month-old (4M), and 7M rats 18 months after irradiation. Cell death and DNA damage response were increased in 4W rats compared to those in controls at 1 month post-irradiation. More Ki-67-positive cells were observed in 4W rats at 12 months post-irradiation. Thyroid tumors were confirmed in 61.9% (13/21), 63.6% (7/11), and 33.3% (2/6) of irradiated 4W, 4M, and 7M rats, respectively, compared to 0%, 14.3% (1/7), and 16.7% (1/6) in the respective nonirradiated controls. There were 29, 9, and 2 tumors in irradiated 4W, 4M, and 7M rats, respectively. The expression of several autophagy components was downregulated in the area surrounding radiation-induced thyroid carcinomas in 4W and 7M rats. LC3 and p62 expression levels decreased in radiation-induced follicular carcinoma in 4W rats. Radiosensitive cells causing thyroid tumors may be more prevalent in young rats, and abrogation of autophagy may be associated with radiation-induced thyroid carcinogenesis.

Reevaluation of the Effect of Iodine on Thyroid Cell Survival and Function Using PCCL3 and Nthy-ori 3-1 Cells.

The appropriate amount of iodine is critical for normal function of thyroid cells synthesizing thyroid hormones. Although normal thyroid cell lines such as rat PCCL3 and FRTL5 and human Nthy-ori 3-1 have been widely used for in vitro studies on physiological and pathophysiological effects of iodine on thyroid cells, we have recently pointed out the critical differences between FRTL5/PCCL3 cells and Nthy-ori 3-1 cells. Therefore, we here directly compared some of the cellular characteristics-iodine uptake, differentiated status, iodine-induced cytotoxicity, and iodine-regulation of autophagy-between PCCL3 and Nthy-ori 3-1 cells. PCCL3 cells express messenger RNAs for thyrotropin receptor and sodium/iodine symporter and incorporate iodine in a thyrotropin-dependent manner, whereas Nthy-ori 3-1 cells do not either. Nevertheless, both cells were comparably resistant to iodine cytotoxicity: Only far excess iodine (5 × 10-2 M) killed 20% to 40% cells in 24 hours with perchlorate exhibiting no effect, suggesting this cytotoxic effect is due to extracellular iodine. In contrast, a wide range of iodine (5 × 10-9 to 5 × 10-2 M) induced autophagy in PCCL3 cells, which was abolished by perchlorate, indicating intracellular iodine-induction of autophagy, but this effect was not observed in Nthy-ori 3-1 cells. In conclusion, it is critical to discriminate the effect of iodine incorporated into cells from that of extracellular iodine on thyroid cells. Iodine-uptake competent thyroid cells such as PCCL3 and FRTL5 cells, not Nthy-ori 3-1 cells, should be used for studies on iodine effect on thyroid cells.

Graves' disease.

Graves' disease (GD) is an autoimmune disease that primarily affects the thyroid gland. It is the most common cause of hyperthyroidism and occurs at all ages but especially in women of reproductive age. Graves' hyperthyroidism is caused by autoantibodies to the thyroid-stimulating hormone receptor (TSHR) that act as agonists and induce excessive thyroid hormone secretion, releasing the thyroid gland from pituitary control. TSHR autoantibodies also underlie Graves' orbitopathy (GO) and pretibial myxoedema. Additionally, the pathophysiology of GO (and likely pretibial myxoedema) involves the synergism of insulin-like growth factor 1 receptor (IGF1R) with TSHR autoantibodies, causing retro-orbital tissue expansion and inflammation. Although the aetiology of GD remains unknown, evidence indicates a strong genetic component combined with random potential environmental insults in an immunologically susceptible individual. The treatment of GD has not changed substantially for many years and remains a choice between antithyroid drugs, radioiodine or surgery. However, antithyroid drug use can cause drug-induced embryopathy in pregnancy, radioiodine therapy can exacerbate GO and surgery can result in hypoparathyroidism or laryngeal nerve damage. Therefore, future studies should focus on improved drug management, and a number of important advances are on the horizon.

Hormonal Regulation of Autophagy in Thyroid PCCL3 Cells and the Thyroids of Male Mice.

Autophagy is an evolutionarily conserved catabolic process by which cells degrade intracellular proteins and organelles in the lysosomes and recycle their metabolites. We have recently demonstrated the crucial role for the basal level of autophagic activity in thyrocyte survival and homeostasis using the thyroid-specific autophagy knockout mice. Here, we first studied hormonal regulation of autophagy in thyrocytes in vitro using a rat thyroid cell line PCCl3 and in vivo with mice. In cultured PCCl3 cells, thyroxine decreased microtubule-associated protein 1 light chain 3 (LC3) puncta (a component of autophagosome) and increased p62 (an autophagy substrate) levels, showing thyroxine-suppression of autophagy. In contrast, TSH increased both LC3 puncta and p62 levels, but at the same time stabilized p62 protein by inhibiting p62 degradation, indicating TSH induction of autophagy. Our experiments with various inhibitors identified that both the cAMP-protein kinase (PK) A-cAMP response element binding protein/ERK and PKC signaling pathways regulates positively autophagic activity. The in vivo results obtained with wild-type mice treated with methimazole and perchlorate or thyroxine were consistent with in vitro results. Next, in thyroid-specific autophagy knockout mice treated with methimazole and perchlorate (that is, mice were placed under a stressed condition where enhanced autophagy was required) for 2 months, lower follicle sizes and lower thyroglobulin contents in thyrocytes were observed, suggesting impaired thyroglobulin production presumably from insufficient nutrient supply. We therefore conclude that TSH positively regulates autophagic activity through the cAMP-PKA-cAMP response element binding protein/ERK and PKC signaling pathways, whereas thyroid hormones inhibit its activity in thyrocytes. Metabolites produced by autophagy appear to be necessary for protein synthesis stimulated by TSH.

Causative role for defective expression of mitochondria-eating protein in accumulation of mitochondria in thyroid oncocytic cell tumors.

Oncocytic cell tumor of the thyroid is composed of large polygonal cells with eosinophilic cytoplasm that is rich in mitochondria. These tumors frequently have the mutations in mitochondrial DNA encoding the mitochondrial electron transport system complex I. However, the mechanism for accumulation of abnormal mitochondria is unknown. A noncanonical mitophagy system has recently been identified, and mitochondria-eating protein (MIEAP) plays a key role in this system. We therefore hypothesized that accumulation of abnormal mitochondria could be attributed to defective MIEAP expression in these tumors. We first show that MIEAP was expressed in all the conventional thyroid follicular adenomas (FAs)/adenomatous goiters (AGs) but not in oncocytic FAs/AGs; its expression was defective not only in oncocytic thyroid cancers but also in the majority of conventional thyroid cancers. Expression of MIEAP was not correlated with methylation status of the 5'-UTR of the gene. Our functional analysis showed that exogenously induced MIEAP, but not PARK2, reduced the amounts of abnormal mitochondria, as indicated by decreased reactive oxygen species levels, mitochondrial DNA / nuclear DNA ratios, and cytoplasmic acidification. Therefore, together with previous studies showing that impaired mitochondrial function triggers compensatory mitochondrial biogenesis that causes an increase in the amounts of mitochondria, we conclude that, in oncocytic cell tumors of the thyroid, increased abnormal mitochondria cannot be efficiently eliminated because of a loss of MIEAP expression, ie impaired MIEAP-mediated noncanonical mitophagy.

Acceleration of BRAFV600E-induced thyroid carcinogenesis by TGFß signal deficiency in mice.

PURPOSE: Transforming growth factor-ß (TGFß) has pleiotropic actions, including both anti- and pro-tumorigenic abilities. We have previously shown no tumor development in the thyroid-specific TGFß receptor type II knockout (Tgfßr2 KO) mice, indicating the insufficiency of defective TGFß signal itself for thyroid cancer initiation. In the current study, we evaluated whether defective TGFß signal accelerates BRAFV600E-mediated thyroid carcinogenesis in our mouse model, in which intrathyroidal injection of adenovirus expressing Cre under thyroglobulin (TG) promoter (Ad-TgP-Cre) into thyroid lobes of conditional BrafV600E knock-in mice (BrafCA) induces thyroid cancers 12 months later. METHODS: BrafCA/wt;Tgfbr2floxE2/floxE2 mice were generated by crossing Tgfbr2floxE2/floxE2 and BrafCA mice, and Ad-TgP-Cre was injected into the left lobes of 4-6-week-old mice. Mice were sacrificed at 6 and 12 months, and the thyroid tissues were subjected to H&E and immune-histochemistry and -fluorecence. RESULTS: Thyroid tumors were observed in 8 of 10 mice at 6 months and 4 of 7 mice at 12 months. These tumors were judged to be malignant by H&E staining, because of the presence of papillary growth of atypical follicular cells, intranuclear cytoplasmic inclusions and so on. Immunohistochemical analyses using thyroid cancer tissues obtained at 6 months demonstrated variable levels of TG but steady levels of Paired Box-8 expression and higher Ki67 positivity. The degree of epithelial-to-mesenchymal transition could not be evaluated because normal thyroid tissues and thyroid cancers developed in BrafCA and BrafCA/wt;Tgfbr2floxE2/floxE2 mice were all E-cadherin+/vimentin-, that is, epithelial type. CONCLUSION: In a mouse model, defective TGFß signaling pathway accelerates BRAFV600E-induced thyroid cancer development, which is occasionally accompanied by reduced TG expression implying dedifferentiation. The former finding is consistent with anti-tumorigenic ability of TGFß in early tumorigenic process, but the latter is contradictory to generally accepted concept for TGFß-induction of dedifferentiation.

Heterogenous nature of gene expression patterns in BRAF-like papillary thyroid carcinomas with BRAFV600E.

PURPOSE: Papillary thyroid cancers (PTCs) are the most common type of thyroid cancers, in which BRAFV600E is the most prevalent driver mutation. It is known that BRAFV600E-positive PTCs are clinically and molecularly heterogenous in terms of aggressiveness and prognosis. The molecular mechanisms of this heterogeneity were evaluated. METHODS: The publicly available RNA-seq data for 26 classical (c) and 5 follicular variant (fv) PTCs with BRAFV600E mutation and the BRAF-like expression signature in the BRAFV600E-RAS score (BRS) and their respective normal adjacent tissues were downloaded, and analyzed for differentially expressed genes (DEGs). The DEGs were then analyzed with the Gene Ontology annotation and the KEGG pathway dataset. RESULTS: We found four lines of evidence for heterogeneity of cPTCs. First, the cluster dendrogram and principle component analyses could not completely distinguish the cancer tissues from normal tissues. Second, the DEGs identified in each sample were highly diverse from one another. Third, although the DEGs were enriched in many terms containing the word "extracellular" ("extracellular region", "extracellular space", and so on) when analyzed as groups, the degree of this enrichment was variable when analyzed individually. Fourth, there are only a few intersections in the over-/underexpressed genes annotated with the terms containing the word "extracellular" among the samples examined. Essentially same results were obtained with BRAF-like, fvPTCs with BRAFV600E. Nevertheless, some frequently over-/underexpressed genes were detected, of which LIPH (lipase H) expression was found to be prognostic and its high expression was favorable for PTCs. CONCLUSION: Groups of BRAF-like, BRAFV600E-positive cPTCs and fvPTCs that are homogenous in regard to histopathology, driver mutation and BRS were found to be highly heterogenous in terms of gene expression patterns. Yet, among the genes that were annotated with the terms containing the word "extracellular" and frequently over-/underexpressed, LIPH is a favorable prognostic marker for PTCs.

Basal Autophagy Deficiency Causes Thyroid Follicular Epithelial Cell Death in Mice.

Autophagy is a catabolic process that involves the degradation of cellular components through the lysosomal machinery, relocating nutrients from unnecessary processes to more pivotal processes required for survival. It has been reported that systemic disruption of the Atg5 or Atg7 gene, a component of autophagy, is lethal and that its tissue-specific disruption causes tissue degeneration in several organs. However, the functional significance of autophagy in the thyroid glands remains unknown. Our preliminary data imply the possible involvement of dysfunctional autophagy in radiation-induced thyroid carcinogenesis. Therefore, we evaluated the effect of Atg5 gene knockout (KO) on thyroid morphology and function. To this end, Atg5flox/flox mice were crossed with TPO-Cre mice, yielding the thyroid follicular epithelial cell (thyrocyte)‒specific ATG5-deficient mice (Atg5thyr-KO/KO). Atg5 gene KO was confirmed by a lack of ATG5 expression, and disruption of autophagy was demonstrated by a decrease in microtubule-associated protein 1 light chain 3-II puncta and an increase in p62. Atg5thyr-KO/KO mice were born normally, and thyroid morphology, thyroid weights, and serum T4 and TSH levels were almost normal at 4 months. However, at 8 and 12 months, a decrease in the number of thyrocytes and an increase in TUNEL+-thyrocytes were observed in Atg5thyr-KO/KO mice even though thyroid function was still normal. The number of irregularly shaped (gourd-shaped) follicles was also increased. Excess oxidative stress was indicated by increased 8-hydroxy-2'-deoxyguanosine and 53BP1 foci in Atg5thyr-KO/KO mice. These data demonstrate that thyrocytes gradually undergo degradation/cell death in the absence of basal levels of autophagy, indicating that autophagy is critical for the quality control of thyrocytes.

Mouse models of sporadic thyroid cancer derived from BRAFV600E alone or in combination with PTEN haploinsufficiency under physiologic TSH levels.

The BRAFV600E mutation is the most prevalent driver mutation of sporadic papillary thyroid cancers (PTC). It was previously shown that prenatal or postnatal expression of BRAFV600E under elevated TSH levels induced thyroid cancers in several genetically engineered mouse models. In contrast, we found that postnatal expression of BRAFV600E under physiologic TSH levels failed to develop thyroid cancers in conditional transgenic Tg(LNL-BrafV600E) mice injected in the thyroid with adenovirus expressing Cre under control of the thyroglobulin promoter (Ad-TgP-Cre). In this study, we first demonstrated that BrafCA/+ mice carrying a Cre-activated allele of BrafV600E exhibited higher transformation efficiency than Tg(LNL-BrafV600E) mice when crossed with TPO-Cre mice. As a result, most BrafCA/+ mice injected with Ad-TgP-Cre developed thyroid cancers in 1 year. Histologic examination showed follicular or cribriform-like structures with positive TG and PAX staining and no colloid formation. Some tumors also had papillary structure component with lower TG expression. Concomitant PTEN haploinsufficiency in injected BrafCA/+;Ptenf/+ mice induced tumors predominantly exhibiting papillary structures and occasionally undifferentiated solid patterns with normal to low PAX expression and low to absent TG expression. Typical nuclear features of human PTC and extrathyroidal invasion were observed primarily in the latter mice. The percentages of pERK-, Ki67- and TUNEL-positive cells were all higher in the latter. In conclusion, we established novel thyroid cancer mouse models in which postnatal expression of BRAFV600E alone under physiologic TSH levels induces PTC. Simultaneous PTEN haploinsufficiency tends to promote tumor growth and de-differentiation.

Thyroid Autoimmunity and Thyroid Cancer - The Pathogenic Connection: A 2018 Update.

The association between thyroid cancer and thyroid autoimmunity has long been suggested, but remains to be elucidated for several decades. Here the data on this issue are updated by summarizing relevant papers published between 2012 and early 2018. Although numerous papers demonstrated the significant increase in the prevalence of thyroid autoimmunity (positive intrathyroidal lymphocyte infiltration and/or anti-thyroglobulin/thyroid peroxidase antibodies) in patients with thyroid cancers as compared to those with benign nodules, and also the significant increase in the prevalence of papillary thyroid cancer (PTC) in patients with thyroid autoimmunity as compared to those without, there are some crucial biases that should be taken into account for their interpretation. However, a difference in the incidence of thyroid autoimmunity in patients with PTCs and those with other types of thyroid cancers appears to support the significant association of two conditions. Thyroid autoimmunity is, at least partly, likely to be elicited against antigens shared by normal and cancerous thyroid tissues, thereby inducing autoimmunity. At the same time, elevated TSH levels (even within the normal reference ranges), which often accompany Hashimoto's patients are a risk factor for thyroid cancer. However, it is still unclear whether or not the co-existence of thyroid autoimmunity impacts on cancer characteristics and prognosis. This issue needs to be further investigated with large-scale prospective studies.

Intracellular redox status controls spherogenicity, an in vitro cancer stem cell marker, in thyroid cancer cell lines.

Cancer stem cells (CSCs), a small fraction of a tumor mass, are proposed to be highly crucial for cancer initiation, recurrence and metastasis. We have recently found that aldehyde dehydrogenase (ALDH) 1A3 is a CSC marker in some thyroid cancer cell lines, whose functional activity is, however, not relevant for thyroid cancer stemness. Since previous studies on malignancies in other organs suggest that intracellular reactive oxygen species (ROS) might be a functional and targetable CSC marker, the present study was conducted to elucidate the significance of ROS as a functional CSC marker in thyroid cancer cell lines. We first found that ROS levels controlled spherogenicity; that is, ROSlow cells were more spherogenic than ROShigh cells. However, unlike typical CSCs in other cancers, CSC-like ROSlow cells in thyroid cancer cells were plastic and were not accompanied by de-differentiation status (i.e., expression of stemness markers/thyroid-specific transcription factors) or chemo-/radio-resistance. The lower levels of ROS were functionally critical because a forced increase in ROS levels by L-buthionine-S,R-sulfoximine, an inhibitor of glutathione (GSH) synthesis, and irradiation suppressed spherogenicity. ROS levels were also correlated with the number of double strand DNA breaks determined by 53BP1 staining. Lower ROS levels appear to be a result of decreased mitochondrial oxidative phosphorylation and elevated GSH contents. Given the importance of CSC-targeted therapy for achieving long-term disease eradication by exhausting self-renewal and growth potential of cancer tissues, ROS may be a good candidate for CSC-targeted therapy in thyroid cancer.

Radiation-related thyroid autoimmunity and dysfunction.

The thyroid gland is vulnerable not only to external radiation but also to internal radiation, because the thyroid cells can incorporate radioactive iodine when synthesizing thyroid hormones. Since radiation-induction of thyroid neoplasia, including thyroid cancer, is well recognized, the data on radiation-related thyroid autoimmunity and dysfunction are summarized and reviewed. High-dose irradiation, irrespective of being external or internal, is strongly associated with a risk of hypothyroidism (with the prevalence ranging from 2.4% to 31%) and of Graves' hyperthyroidism (with the prevalence being up to 5%). It is easy to understand that high-dose irradiation induces hypothyroidism with some frequency, because high-dose irradiation destroys the thyroid gland. On the other hand, the basis for development of hyperthyroidism is mechanistically unclear, and it is merely speculative that autoantigens may be released from damaged thyroid glands and recognized by the immune system, leading to the development of anti-thyrotropin receptor antibodies and Graves' hyperthyroidism in subjects who are immunologically predisposed to this ailment. In contrast, the data on moderate to low-dose irradiation on thyroid autoimmunity and dysfunction are inconsistent. Although it is difficult to draw a definitive conclusion, some data may suggest a transient effect of moderate- to low-dose irradiation on hypothyroidism and autoimmune thyroiditis, implying that the effect, if it exists, is reversible. Finally, no report has shown a statistically significant increase in the prevalence of moderate- to low-dose irradiation-induced Graves' hyperthyroidism.