An Orally Efficacious Thyrotropin Receptor Ligand Inhibits Growth and Metastatic Activity of Thyroid Cancers.

CONTEXT: Thyroid-stimulating hormone (or thyrotropin) receptor (TSHR) could be a selective target for small molecule ligands to treat thyroid cancer (TC). OBJECTIVE: We report a novel, orally efficacious ligand for TSHR that exhibits proliferation inhibitory activity against human TC in vitro and in vivo, and inhibition of metastasis in vivo. DESIGN: A35 (NCATS-SM4420; NCGC00241808) was selected from a sub-library of >200 TSHR ligands. Cell proliferation assays including BrdU incorporation and WST-1, along with molecular docking studies were done. In vivo activity of A35 was assessed in TC cell-derived xenograft (CDX) models with immunocompromised (NSG) mice. FFPE sections of tumor and lung tissues were observed for the extent of cell death and metastasis. RESULTS: A35 was shown to stimulate cAMP production in some cell types by activating TSHR but not in TC cells, MDA-T32 and MDA-T85. A35 inhibited proliferation of MDA-T32 & MDA-T85 in vitro and in vivo, and pulmonary metastasis of MDA-T85F1 in mice. In vitro, A35 inhibition of proliferation was reduced by a selective TSHR antagonist. Inhibition of CDX tumor growth without decreases in mouse weights and liver function showed A35 to be efficacious without apparent toxicity. Lastly, A35 reduced levels of Ki67 in the tumors and metastatic markers in lung tissues. CONCLUSION: We conclude that A35 is a TSHR-selective inhibitor of TC cell proliferation and metastasis, and suggest that A35 may be a promising lead drug candidate for the treatment of differentiated thyroid cancer in humans.

Proximity ligation assay to study TSH receptor homodimerization and crosstalk with IGF-1 receptors in human thyroid cells.

Proximity ligation assay (PLA) is a methodology that permits detection of protein-protein closeness, that is, proteins that are within 40 nanometers of each other, in cells or tissues at endogenous protein levels or after exogenous overexpression. It detects the protein(s) with high sensitivity and specificity because it employs a DNA hybridization step followed by DNA amplification. PLA has been used successfully with many types of proteins. In this methods paper, we will describe the workings of PLA and provide examples of its use to study TSH/IGF-1 receptor crosstalk in Graves' orbital fibroblasts (GOFs) and TSH receptor homodimerization in primary cultures of human thyrocytes.

Opposing Effects of EGF Receptor Signaling on Proliferation and Differentiation Initiated by EGF or TSH/EGF Receptor Transactivation.

Regulation of thyroid cells by thyrotropin (TSH) and epidermal growth factor (EGF) has been known but different effects of these regulators on proliferation and differentiation have been reported. We studied these responses in primary cultures of human thyroid cells to determine whether TSH receptor (TSHR) signaling may involve EGF receptor (EGFR) transactivation. We confirm that EGF stimulates proliferation and de-differentiation whereas TSH causes differentiation in the absence of other growth factors. We show that TSH/TSHR transactivates EGFR and characterize it as follows: (1) TSH-induced upregulation of thyroid-specific genes is inhibited by 2 inhibitors of EGFR kinase activity, AG1478 and erlotinib; (2) the mechanism of transactivation is independent of an extracellular EGFR ligand by showing that 2 antibodies, cetuximab and panitumumab, that completely inhibited binding of EGFR ligands to EGFR had no effect on transactivation, and by demonstrating that no EGF was detected in media conditioned by thyrocytes incubated with TSH; (3) TSH/TSHR transactivation of EGFR is different than EGFR activation by EGF by showing that EGF led to rapid phosphorylation of EGFR whereas transactivation occurred in the absence of receptor phosphorylation; (4) EGF caused downregulation of EGFR whereas transactivation had no effect on EGFR level; (5) EGF and TSH stimulation converged on the protein kinase B (AKT) pathway, because TSH, like EGF, stimulated phosphorylation of AKT that was inhibited by EGFR inhibitors; and (6) TSH-induced upregulation of thyroid genes was inhibited by the AKT inhibitor MK2206. Thus, TSH/TSHR causes EGFR transactivation that is independent of extracellular EGFR ligand and in part mediates TSH regulation of thyroid hormone biosynthetic genes.

Graves' Autoantibodies Exhibit Different Stimulating Activities in Cultures of Thyrocytes and Orbital Fibroblasts Not Reflected by Clinical Assays.

Background: The pathogenesis of Graves' hyperthyroidism (GH) and associated Graves' orbitopathy (GO) appears to involve stimulatory autoantibodies (thyrotropin receptor [TSHR]-stimulating antibodies [TSAbs]) that bind to and activate TSHRs on thyrocytes and orbital fibroblasts. In general, measurement of circulating TSHR antibodies by clinical assays correlates with the status of GH and GO. However, most clinical measurements of TSHR antibodies use competitive binding assays that do not distinguish between TSAbs and antibodies that bind to but do not activate TSHRs. Moreover, clinical assays for TSAbs measure stimulation of only one signaling pathway, the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway, in engineered cells that are not thyrocytes or orbital fibroblasts. We determined whether measuring TSAbs by a cAMP-PKA readout in engineered cells accurately reveals the efficacies of stimulation by these antibodies on thyrocytes and orbital fibroblasts. Methods: We measured TSAb stimulation of normal human thyrocytes and orbital fibroblasts from patients with GO in primary cultures in vitro. In thyrocytes, we measured secretion of thyroglobulin (TG) and in orbital fibroblasts secretion of hyaluronan (hyaluronic acid [HA]). We also measured stimulation of cAMP production in engineered TSHR-expressing cells in an assay similar to clinical assays. Furthermore, we determined whether there were differences in stimulation of thyrocytes and orbital fibroblasts by TSAbs from patients with GH alone versus from patients with GO understanding that patients with GO have accompanying GH. Results: We found a positive correlation between TSAb stimulation of cAMP production in engineered cells and TG secretion by thyrocytes as well as HA secretion by orbital fibroblasts. However, TSAbs from GH patients stimulated thyrocytes more effectively than TSAbs from GO patients, whereas TSAbs from GO patients were more effective in activating orbital fibroblasts than TSAbs from GH patients. Conclusions: Clinical assays of stimulation by TSAbs measuring activation of the cAMP-PKA pathway do correlate with stimulation of thyrocytes and orbital fibroblasts; however, they do not distinguish between TSAbs from GH and GO patients. In vitro, TSAbs exhibit selectivity in activating TSHRs since TSAbs from GO patients were more effective in stimulating orbital fibroblasts and TSAbs from GH patients were more effective in stimulating thyrocytes.

TSH stimulation of human thyroglobulin and thyroid peroxidase gene transcription is partially dependent on internalization.

The TSH receptor (TSHR) is the major regulator of thyroid hormone biosynthesis in human thyrocytes by regulating the transcription of a number of genes including thyroglobulin (TG) and thyroperoxidase (TPO). Until recently, it was thought that TSHR initiated signal transduction pathways only at the cell-surface and that internalization was primarily involved in TSHR desensitization and downregulation. Studies primarily in mouse cells showed that TSHR internalization regulates gene transcription at an intracellular site also. However, this has not been shown for genes involved in thyroid hormone biosynthesis in human thyrocytes. We used human thyrocytes in primary culture. In these cells, the dose-response to TSH for gene expression is biphasic with low doses upregulating gene expression and higher doses decreasing gene expression. We used two approaches to inhibit internalization. In the first, we used inhibitors of dynamins, dynasore and dyngo-4a. Pretreatment with dynasore or dyngo-4a markedly inhibited TSH upregulation of TG and TPO mRNAs, as well as TG secretion. In the second, we used knockdown of dynamin 2, which is the most abundant dynamin in human thyrocytes. We showed that dynamin 2 knockdown inhibited TSHR internalization and decreased the TSH-stimulated levels of TG and TPO mRNAs and proteins. Lastly, we showed that the level of the activatory transcription factor phosphorylated cAMP response element binding protein (pCREB) in the cell nuclei was reduced by 68% when internalization was inhibited. We conclude that upregulation of genes involved in thyroid hormone synthesis in human thyrocytes is, in part, dependent on internalization leading to nuclear localization of an activated transcription factor(s).

Inhibition of TSH/IGF-1 Receptor Crosstalk by Teprotumumab as a Treatment Modality of Thyroid Eye Disease.

CONTEXT: We previously presented evidence that TSH receptor (TSHR)-stimulating autoantibodies (TSAbs) bind to and activate TSHRs but do not bind to IGF1 receptors (IGF1Rs). Nevertheless, we showed that IGF1Rs were involved in thyroid eye disease (TED) pathogenesis because TSAbs activated crosstalk between TSHR and IGF1R. Teprotumumab, originally generated to inhibit IGF1 binding to IGF1R, was recently approved for the treatment of TED (Tepezza). OBJECTIVE: To investigate the role of TSHR/IGF1R crosstalk in teprotumumab treatment of TED. DESIGN: We used orbital fibroblasts from patients with TED (TEDOFs) and measured stimulated hyaluronan (HA) secretion as a measure of orbital fibroblast activation by TED immunoglobulins (TED-Igs) and monoclonal TSAb M22. We previously showed that M22, which does not bind to IGF1R, stimulated HA in a biphasic dose-response with the higher potency phase dependent on TSHR/IGF1R crosstalk and the lower potency phase independent of IGF1R. Stimulation by TED-Igs and M22 was measured in the absence or presence of teprotumumab biosimilar (Tepro) or K1-70, an antibody that inhibits TSHR. RESULTS: We show: (1) Tepro dose-dependently inhibits stimulation by TED-Igs; (2) Tepro does not bind to TSHRs; (3) Tepro inhibits IGF1R-dependent M22-induced HA production, which is mediated by TSHR/IGF1R crosstalk, but not IGF1R-independent M22 stimulation; and (4) ß-arrestin 1 knockdown, which blocks TSHR/IGF1R crosstalk and prevents Tepro inhibition of HA production by M22 and by a pool of TED-Igs. CONCLUSION: We conclude that Tepro inhibits HA production by TEDOFs by inhibiting TSHR/IGF1R crosstalk and suggest that inhibition of TSHR/IGF1R crosstalk is the mechanism of its action in treating TED.

Is There Evidence for IGF1R-Stimulating Abs in Graves' Orbitopathy Pathogenesis?

In this review, we summarize the evidence against direct stimulation of insulin-like growth factor 1 receptors (IGF1Rs) by autoantibodies in Graves' orbitopathy (GO) pathogenesis. We describe a model of thyroid-stimulating hormone (TSH) receptor (TSHR)/IGF1R crosstalk and present evidence that observations indicating IGF1R's role in GO could be explained by this mechanism. We evaluate the evidence for and against IGF1R as a direct target of stimulating IGF1R antibodies (IGF1RAbs) and conclude that GO pathogenesis does not involve directly stimulating IGF1RAbs. We further conclude that the preponderance of evidence supports TSHR as the direct and only target of stimulating autoantibodies in GO and maintain that the TSHR should remain a major target for further development of a medical therapy for GO in concert with drugs that target TSHR/IGF1R crosstalk.

Thyrotropin regulation of differentiated gene transcription in adult human thyrocytes in primary culture.

Thyroid transcription factors (TTFs) - NKX2-1, FOXE1, PAX8 and HHEX - regulate multiple genes involved in thyroid development in mice but little is known about TTF regulation of thyroid-specific genes - thyroglobulin (TG), thyroid peroxidase (TPO), deiodinase type 2 (DIO2), sodium/iodide symporter (NIS) and TSH receptor (TSHR) - in adult, human thyrocytes. Thyrotropin (thyroid-stimulating hormone, TSH) regulation of thyroid-specific gene expression in primary cultures of human thyrocytes is biphasic yielding an inverted U-shaped dose-response curve (IUDRC) with upregulation at low doses and decreases at high doses. Herein we show that NKX2-1, FOXE1 and PAX8 are required for TSH-induced upregulation of the mRNA levels of TG, TPO, DIO2, NIS, and TSHR whereas HHEX has little effect on the levels of these thyroid-specific gene mRNAs. We show that TSH-induced upregulation is mediated by changes in their transcription and not by changes in the degradation of their mRNAs. In contrast to the IUDRC of thyroid-specific genes, TSH effects on the levels of the mRNAs for NKX2-1, FOXE1 and PAX8 exhibit monophasic decreases at high doses of TSH whereas TSH regulation of HHEX mRNA levels exhibits an IUDRC that overlaps the IUDRC of thyroid-specific genes. In contrast to findings during mouse development, TTFs do not have major effects on the levels of other TTF mRNAs in adult, human thyrocytes. Thus, we found similarities and important differences in the regulation of thyroid-specific genes in mouse development and TSH regulation of these genes in adult, human thyrocytes.

ß-Arrestin 1 in Thyrotropin Receptor Signaling in Bone: Studies in Osteoblast-Like Cells.

A direct action of thyrotropin (TSH, thyroid-stimulating hormone) on bone precursors in humans is controversial. Studies in rodent models have provided conflicting findings. We used cells derived from a moderately differentiated osteosarcoma stably overexpressing human TSH receptors (TSHRs) as a model of osteoblast precursors (U2OS-TSHR cells) to investigate TSHR-mediated effects in bone differentiation in human cells. We review our findings that (1) TSHR couples to several different G proteins to induce upregulation of genes associated with osteoblast activity-interleukin 11 (IL-11), osteopontin (OPN), and alkaline phosphatase (ALPL) and that the kinetics of the induction and the G protein-mediated signaling pathways involved were different for these genes; (2) TSH can stimulate ß-arrestin-mediated signal transduction and that ß-arrestin 1 in part mediates TSH-induced pre-osteoblast differentiation; and (3) TSHR/insulin-like growth factor 1 (IGF1) receptor (IGF1R) synergistically increased OPN secretion by TSH and IGF1 and that this crosstalk was mediated by physical association of these receptors in a signaling complex that uses ß-arrestin 1 as a scaffold. These findings were complemented using a novel ß-arrestin 1-biased agonist of TSHR. We conclude that TSHR can signal via several transduction pathways leading to differentiation of this model system of human pre-osteoblast cells and, therefore, that TSH can directly regulate these bone cells.

TSH Receptor Homodimerization in Regulation of cAMP Production in Human Thyrocytes in vitro.

Thyrotropin hormone (TSH) was reported to exhibit biphasic regulation of cAMP production in human thyroid slices; specifically, upregulation at low TSH doses transitioning to inhibition at high doses. We observed this phenomenon in HEK293 cells overexpressing TSH receptors (TSHRs) but in only 25% of human thyrocytes (hThyros) in vitro. Because TSHR expression in hThyros in vitro was low, we tested the hypothesis that high, in situ levels of TSHRs were needed for biphasic cAMP regulation. We increased expression of TSHRs by infecting hThyros with adenoviruses expressing human TSHR (AdhTSHR), measured TSH-stimulated cAMP production and TSHR homodimerization. TSHR mRNA levels in hThyros in vitro were 100-fold lower than in human thyroid tissue. AdhTSHR infection increased TSHR mRNA expression to levels found in thyroid tissue and flow cytometry showed that cell-surface TSHRs increased more than 15-fold. Most uninfected hThyro preparations exhibited monotonic cAMP production. In contrast, most hThyro preparations infected with AdhTSHR expressing TSHR at in vivo levels exhibited biphasic TSH dose responses. Treatment of AdhTSHR-infected hThyros with pertussis toxin resulted in monotonic dose response curves demonstrating that lower levels of cAMP production at high TSH doses were mediated by Gi/Go proteins. Proximity ligation assays confirmed that AdhTSHR infection markedly increased the number of TSHR homodimers. We conclude that in situ levels of TSHRs as homodimers are needed for hThyros to exhibit biphasic TSH regulation of cAMP production.

TSH/IGF1 receptor crosstalk: Mechanism and clinical implications.

Increasing evidence of interdependence between G protein-coupled receptors and receptor tyrosine kinase signaling pathways has prompted reevaluation of crosstalk between these receptors in disease and therapy. Investigations into thyroid-stimulating hormone (TSH) and insulin-like growth factor 1 (IGF1) receptor crosstalk, and its application to the clinic have in particular shown recent progress. In this review, we summarize current insights into the mechanism of TSH/IGF1 receptor crosstalk. We discuss evidence that crosstalk is one of the underlying causes of TSHR-based disease and the feasibility of using combinations of TSH receptor and IGF1 receptor antagonists to increase the therapeutic index for the treatment of Graves' hyperthyroidism and Graves' ophthalmopathy.

Targeting TSH and IGF-1 Receptors to Treat Thyroid Eye Disease.

Graves' disease (GD) is an autoimmune disease caused in part by thyroid-stimulating antibodies (TSAbs) that activate the thyroid-stimulating hormone receptor (TSHR). In Graves' hyperthyroidism (GH), TSAbs cause persistent stimulation of thyroid cells leading to continuous thyroid hormone synthesis and secretion. Thyroid eye disease (TED), also called Graves' orbitopathy, is an orbital manifestation of GD. We review the important roles of the TSHR and the insulin-like growth factor 1 receptor (IGF-1R) in the pathogenesis of TED and discuss a model of TSHR/IGF-1R crosstalk that considers two pathways initiated by TSAb activation of TSHR in the eye, an IGF-1R-independent and an IGF-1R-dependent signaling pathway leading to hyaluronan (HA) secretion in orbital fibroblasts. We discuss current and future therapeutic approaches targeting the IGF-1R and TSHR. Teprotumumab, a human monoclonal anti-IGF-1R-blocking antibody, has been approved as an effective treatment in patients with TED. However, as the TSHR seems to be the primary target for TSAbs in patients with GD, future therapeutic interventions directly targeting the TSHR, e.g. blocking antibodies and small molecule antagonists, are being developed and have the advantage to inhibit the IGF-1R-independent as well as the IGF-1R-dependent component of TSAb-induced HA secretion. Antigen-specific immunotherapies using TSHR peptides to reduce serum TSHR antibodies are being developed also. These TSHR-targeted strategies also have the potential to treat both GH and TED with the same drug. We propose that combination therapy targeting TSHR and IGF-1R may be an effective and better tolerated treatment strategy for TED.

Thyrotropin, but Not Thyroid-Stimulating Antibodies, Induces Biphasic Regulation of Gene Expression in Human Thyrocytes.

Background: Thyrotropin (TSH) and thyroid-stimulating antibodies (TSAbs) activate TSH receptor (TSHR) signaling by binding to its extracellular domain. TSHR signaling has been studied extensively in animal thyrocytes and in engineered cell lines, and differences in signaling have been observed in different cell systems. We, therefore, decided to characterize and compare TSHR signaling mediated by TSH and monoclonal TSAbs in human thyrocytes in primary culture. Methods: We used quantitative reverse transcription-polymerase chain reaction to measure mRNA levels of thyroid-specific genes thyroglobulin (TG), thyroperoxidase (TPO), iodothyronine deiodinase type 2 (DIO2), sodium-iodide symporter (NIS), and TSHR after stimulation by TSH or two monoclonal TSAbs, KSAb1 and M22. We also compared secreted TG protein after TSHR activation by TSH and TSAbs using an enzyme-linked immunosorbent assay. TSHR cell surface expression was determined using fluorescence activated cell sorting (FACS). Results: We found that TSH at low doses increases and at high doses (>1 mU/mL) decreases levels of gene expression for TSHR, TG, TPO, NIS, and DIO2. The biphasic effect of TSH on signaling was not caused by downregulation of cell surface TSHRs. This bell-shaped biphasic dose-response curve has been termed an inverted U-shaped dose-response curve (IUDRC). An IUDRC was also found for TSH-induced regulation of TG secretion. In contrast, KSAb1- and M22-induced regulation of TSHR, TG, TPO, NIS, and DIO2 gene expression, and secreted TG followed a monotonic dose-response curve that plateaus at high doses of activating antibody. Conclusions: Our data demonstrate that the physiological activation of TSHRs by TSH in primary cultures of human thyrocytes is characterized by a regulatory mechanism that may inhibit thyrocyte overstimulation. In contrast, TSAbs do not exhibit biphasic regulation. Although KSAb1 and M22 may not be representative of all TSAbs found in patients with Graves' disease, we suggest that persistent robust stimulation of TSHRs by TSAbs, unrelieved by a decrease at high TSAb levels, fosters chronic stimulation of thyrocytes in Graves' hyperthyroidism.

Thyrotropin Causes Dose-dependent Biphasic Regulation of cAMP Production Mediated by Gs and Gi/o Proteins.

The thyrotropin (TSH) receptor (TSHR) signals via G proteins of all four classes and ß-arrestin 1. Stimulation of TSHR leads to increasing cAMP production that has been reported as a monotonic dose-response curve that plateaus at high TSH doses. In HEK 293 cells overexpressing TSHRs (HEK-TSHR cells), we found that TSHR activation exhibits an "inverted U-shaped dose-response curve" with increasing cAMP production at low doses of TSH and decreased cAMP production at high doses (>1 mU/ml). Since protein kinase A inhibition by H-89 and knockdown of ß-arrestin 1 or ß-arrestin 2 did not affect the decreased cAMP production at high TSH doses, we studied the roles of TSHR downregulation and of Gi/Go proteins. A high TSH dose (100 mU/ml) caused a 33% decrease in cell-surface TSHR. However, because inhibiting TSHR downregulation with combined expression of a dominant negative dynamin 1 and ß-arrestin 2 knockdown had no effect, we concluded that downregulation is not involved in the biphasic cAMP response. Pertussis toxin, which inhibits activation of Gi/Go, abolished the biphasic response with no statistically significant difference in cAMP levels at 1 and 100 mU/ml TSH. Concordantly, co-knockdown of Gi/Go proteins increased cAMP levels stimulated by 100 mU/ml TSH from 55% to 73% of the peak level. These data show that biphasic regulation of cAMP production is mediated by Gs and Gi/Go at low and high TSH doses, respectively, which may represent a mechanism to prevent overstimulation in TSHR-expressing cells. SIGNIFICANCE STATEMENT: We demonstrate biphasic regulation of TSH-mediated cAMP production involving coupling of the TSH receptor (TSHR) to Gs at low TSH doses and to Gi/o at high TSH doses. We suggest that this biphasic cAMP response allows the TSHR to mediate responses at lower levels of TSH and that decreased cAMP production at high doses may represent a mechanism to prevent overstimulation of TSHR-expressing cells. This mechanism could prevent chronic stimulation of thyroid gland function.

Arrestin-ß-1 Physically Scaffolds TSH and IGF1 Receptors to Enable Crosstalk.

Endogenously expressed TSH receptors (TSHRs) on orbital fibroblasts of patients with Graves ophthalmopathy (GO) use crosstalk with IGF1 receptors (IGF1R) to synergistically stimulate secretion of hyaluronan (HA), a major component of GO pathology. We previously showed crosstalk occurred upstream of mitogen-activated protein kinase (ERK) phosphorylation. Because other G protein-coupled receptors engage arrestin-ß-1 (ARRB1) and ERK, we tested whether ARRB1 was a necessary component of TSHR/IGF1R crosstalk. HA secretion was stimulated by the TSHR-stimulating monoclonal antibodies M22 and KSAb1, or immunoglobulins from patients with GO (GO-Igs). Treatment with M22, as previously shown, resulted in biphasic dose-response stimulation of HA secretion. The high-potency phase was IGF1R dependent, and the low-potency phase was partly IGF1R independent. KSAb1 produced a monophasic dose-response stimulation of HA secretion, whose potency was lowered >20-fold after IGF1R knockdown. ARRB1 knockdown abolished M22's high-potency phase and lowered KSAb1's potency and efficacy. ARRB1 knockdown inhibited GO-Ig stimulation of HA secretion and of ERK phosphorylation. Last, ARRB1 was shown to be necessary for TSHR/IGF1R proximity. In contrast, ARRB2 knockdowns did not show these effects. Thus, TSHR must neighbor IGF1R for crosstalk in GO fibroblasts to occur, and this depends on ARRB1 acting as a scaffold. Similar scaffolding of TSHR and IGF1R by ARRB1 was found in human osteoblast-like cells and human thyrocytes. These findings support a model of TSHR/IGF1R crosstalk that may be a general mechanism for G-protein-coupled receptor/receptor tyrosine kinase crosstalk dependent on ARRB1.

Association of Thyrotropin Suppression With Survival Outcomes in Patients With Intermediate- and High-Risk Differentiated Thyroid Cancer.

Importance: Suppression of thyrotropin (often referred to as thyroid-stimulating hormone, or TSH) with levothyroxine used in management of intermediate- and high-risk differentiated thyroid cancer (DTC) to reduce the likelihood of progression and death is based on conflicting evidence. Objective: To examine a cohort of patients with intermediate- and high-risk DTC to assess the association of thyrotropin suppression with progression-free survival (PFS) and overall survival. Design, Setting, and Participants: This cohort study used a multicenter database analysis including patients from tertiary referral centers and local clinics followed up for a mean (SD) of 7.2 (5.8) years. Patients with DTC treated uniformly with total thyroidectomy and radioactive iodine between January 1, 1979, and March 1, 2015, were included. Among the 1012 patients, 145 patients were excluded due to the lack of longitudinal thyrotropin measurements. Exposures: Levothyroxine therapy to target thyrotropin suppression with dose adjustments based on changing thyrotropin goal. Main Outcomes and Measures: The primary outcome measures were overall survival and PFS. A Cox proportional hazards model was used to assess the contribution of age, sex, tumor size, histology, and lymph node and distant metastases at landmarks 1.5, 3.0, and 5.0 years. The patients were divided into 3 groups based on mean thyrotropin score before each landmark: (1) suppressed thyrotropin, (2) moderately suppressed or low-normal thyrotropin, and (3) low-normal or elevated thyrotropin. Results: Among 867 patients (557 [64.2%] female; mean [SD] age, 48.5 [16.5] years) treated with a median (range) cumulative dose of 151 (30-1600) mCi radioactive iodine, disease progression was observed in 293 patients (33.8%), and 34 patients (3.9%) died; thus, the study was underpowered in death events. Thyrotropin suppression was not associated with improved PFS at landmarks 1.5 (P = .41), 3.0 (P = .51), and 5.0 (P = .64) years. At 1.5 and 3.0 years, older age (hazard ratio [HR], 1.06; 95% CI, 1.03-1.08 and HR, 1.05; 95% CI, 1.01-1.08, respectively), lateral neck lymph node metastases (HR, 4.64; 95% CI, 2.00-10.70 and HR, 4.02; 95% CI, 1.56-10.40, respectively), and distant metastases (HR, 7.54; 95% CI, 3.46-16.50 and HR, 7.10; 95% CI, 2.77-18.20, respectively) were independently associated with subsequent time to progression, while at 5.0 years, PFS was shorter for patients with lateral neck lymph node metastases (HR, 3.70; 95% CI, 1.16-11.90) and poorly differentiated histology (HR, 71.80; 95% CI, 9.80-526.00). Conclusions and Relevance: Patients with intermediate- and high-risk DTC might not benefit from thyrotropin suppression. This study provides the justification for a randomized trial.

Evaluation of the effects of chemotherapy-induced fatigue and pharmacological interventions in multiple mouse behavioral assays.

Fatigue is a common symptom in many diseases and disorders and can reduce quality of life, yet lacks an adequate pharmacological intervention. To identify and develop such interventions, and to better understand fatigue, additional preclinical research is necessary. However, despite numerous mouse behavioral assays reportedly detecting fatigue-like behavior, the assumption that fatigue-like behavior is detected in many assays has not been validated through a cross-assay study. Thus, we modeled fatigue in mice by administering 5-fluorouracil, a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in mice, then evaluated its effects via voluntary wheel running activity (VWRA), locomotor activity in the open field test (OFT), immobility in the forced swim test (FST), and distance run in the treadmill fatigue test (TFT) and treadmill exercise capacity test. Additionally, taltirelin or methylphenidate was administered to alleviate fatigue-like behavior. As a result of 5-fluorouracil treatment, VWRA and the TFT were markedly reduced, indicating fatigue. The OFT, FST, and treadmill exercise capacity test, however, failed to detect fatigue-like behavior. Interestingly, both taltirelin and methylphenidate alleviated fatigue-like behavior in TFT. These data suggest that, of the current assays, only the TFT and VWRA should be expected to detect fatigue-like behavior. Moreover, this study provides additional evidence that taltirelin may provide a novel treatment for chemotherapy-induced fatigue and warrants further evaluation as an anti-fatigue therapeutic.