The Histamine 3 Receptor Is Expressed in the Heart and Its Activation Opposes Adverse Cardiac Remodeling in the Angiotensin II Mouse Model.

Histamine is a basic amine stored in mast cells, with its release capable of activating one of four histamine receptors. The histamine 3 receptor (H3R) is known to be cardioprotective during acute ischemia by acting to limit norepinephrine release. However, a recent study reported that myofibroblasts isolated from the infarct zone of rat hearts responded to H3R activation by up-regulating collagen production. Thus, it is necessary to clarify the potential role of the H3R in relation to fibrosis in the heart. We identified that the mouse left ventricle (LV) expresses the H3R. Isolation of mouse cardiac fibroblasts determined that while angiotensin II (Ang II) increased levels of the H3R, these cells did not produce excess collagen in response to H3R activation. Using the Ang II mouse model of adverse cardiac remodeling, we found that while H3R blockade had little effect on cardiac fibrosis, activation of the H3R reduced cardiac fibrosis and macrophage infiltration. These findings suggest that when activated, the H3R is anti-inflammatory and anti-fibrotic in the mouse heart and may be a promising target for protecting against cardiac fibrosis.

TERT promoter mutations are a major indicator of recurrence and death due to papillary thyroid carcinomas.

CONTEXT: TERT promoter mutations have been associated with adverse prognosis in papillary thyroid carcinomas (PTCs). OBJECTIVE: We investigated the association between TERT promoter mutations and survival from PTC. DESIGN: Retrospective observational cohort study. PATIENTS: Eighty consecutive patients with PTC who underwent surgery between 1990 and 2003. MEASUREMENTS: TERT promoter was genotyped in DNA from 80 archival PTCs by Sanger sequencing. Median follow-up was 106 months (range 1-270). Outcomes analysis was stratified according to disease and overall survival status. For each parameter, relative risk (RR) adjusted for age at first surgery and gender was estimated. Both univariate and multivariate analyses were performed using logistic regression, Kaplan-Meier survival analysis and Cox regression models. RESULTS: PTCs from 11 patients (14%) contained either C228T or C250T TERT promoter mutation. TERT mutations were significantly associated with adverse prognostic features such as older age (P = 0·002), male gender (P = 0·01) and Stage IV disease (P = 0·03). Four patients died from PTC during follow-up: 3 patients with TERT mutations (27%) and one without (1·5%). Disease-related mortality rate with or without TERT mutations was 33·7 vs 1·6 per 1000 patient-years respectively, that is 10 (95% CI = 1·0-104·1, P = 0·05) fold higher, after adjustment for age at first surgery and gender. The combination of TERT promoter mutation and BRAF(V) (600E) significantly increased disease-related death risk (P = 0·002). TERT mutations increased expression of a reporter gene in thyroid cells containing BRAF(V) (600E) . CONCLUSIONS: TERT promoter mutations are a major indicator of death due to PTCs. Conversely, absence of TERT mutations portends better survival.

The Association between Lymphocytic Thyroiditis and Papillary Thyroid Cancer Harboring Mutant BRAF: A Systematic Review and Meta-Analysis.

Background: Papillary thyroid cancer (PTC) and lymphocytic thyroiditis (LT) co-occur with a prevalence of about 30%. PTC harboring BRAFV600E (PTC-BRAF) confers a worse prognosis, but it is unclear if LT alters prognostic features and recurrence of PTC. Objective: We compared the prevalence of PTC-BRAF with and without LT. The risk of adverse pathological features in (i) PTC in the presence and absence of BRAF mutation, irrespective of LT status, was compared to (ii) PTC in the presence and absence of LT, irrespective of BRAF status. Methods: We searched PubMed, Embase, and Web of Science Core Collection for observational studies published from 2010 to June 2023 on adult patients with PTC. The search strategy yielded 47 studies with relevant data. Data of baseline characteristics, clinicopathological features, and the quality assessment tool were extracted by two reviewers. The study was registered with PROSPERO (CRD42023437492). Results: Of the 47 studies, 39 studies with a total cohort of 28 143, demonstrated that the odds of PTC-BRAF were significantly lower in the presence of LT compared to its absence (odds ratio [OR] 0.53, 95% confidence interval [CI]: 0.48-0.58, p < 0.00001). In PTC-BRAF patients, there was a positive association of central neck nodal disease (CNND), PTC > 1 cm, extra-thyroidal extension, American Joint Committee on Cancer (AJCC) Stage 3-4, and multifocality with pooled ORs of 1.54 (95% CI: 1.16-2.04), 1.14 (95% CI: 0.82-1.58), 1.66 (95% CI: 1.40-1.97), 1.53 (95% CI: 1.35-1.75), and 1.24 (95% CI: 1.11-1.40) respectively, compared to wild-type PTC, irrespective of LT status. In the same studies, PTC with LT patients had lower pooled ORs of 0.64 (95% CI: 0.51-0.81) for CNND, 0.83 (95% CI: 0.73-0.95) for PTC > 1 cm, 0.71 (95% CI: 0.58-0.86) for ETE, 0.84 (95% CI: 0.75-0.94) for AJCC Stage 3-4 compared to PTC without LT, irrespective of BRAF status. PTC recurrence was not affected by BRAF or LT, with pooled ORs of 1.12 (95% CI: 0.66-1.90, p = 0.67) and 0.60 (95% CI: 0.28-1.30, p = 0.20) respectively. Similar results were seen with recurrence expressed as hazard ratio in this limited data-set. Conclusion: The odds of PTC-BRAF are significantly lower in the presence of LT than without. PTC with LT, irrespective of BRAF status, was significantly associated with better prognostic factors. Further studies are required to evaluate if LT inhibits PTC-BRAF, and whether this is relevant to the role of immunotherapy in advanced thyroid cancer.

Polypharmacy With High Drug Burden Index (DBI) Alters the Gut Microbiome Overriding Aging Effects and Is Reversible With Deprescribing.

Aging, medication use, and global function are associated with changes in the microbiome. However, their interrelationships and changes over time require further characterization. In a longitudinal aging mouse study, we investigated the effects of aging, chronic polypharmacy with a high Drug Burden Index (DBI, measure of total anticholinergic and sedative medication exposure) and gradual cessation (deprescribing) on the microbiome, further exploring any association with global outcomes. Chronic administration of high DBI polypharmacy attenuated the aging-related reduction in alpha diversity, which was not sustained after deprescribing. Beta diversity and LEfSe (Linear discriminant analysis Effect Size) features varied with age, polypharmacy, and deprescribing. Aging with and without polypharmacy shared decreases in Bifidobacteriaceae, Paraprevotellaceae, Bacteroidaceae, and Clostridiaceae, while only aging with polypharmacy showed increased LEfSe features. Microbiome diversity correlated with frailty, nesting, and open field performance. Polypharmacy deprescribing reversed changes that occurred with treatment. However, the microbiome did not recover to its pretreatment composition at 12 months, nor develop the same aging-related changes from 12 to 24 months as the control group. Overall, aging, chronic polypharmacy, and deprescribing differentially affected the diversity and composition of the gut microbiome, which is associated with frailty and function.

A Case Report of Poor Response to Selpercatinib in the Presence of a 632_633 RET Deletion.

Background: Genomic deletions in medullary thyroid cancer (MTC) are rare. Selpercatinib is a highly selective RET inhibitor for treatment of metastatic RET-altered MTC. We report a 35-year-old male with an aggressive metastatic MTC harboring p.632_633del RET that was poorly responsive to RET kinase inhibitor selpercatinib. Objective: Our objective was to understand the clinical phenotype of p.632_633del RET in MTC in the context of novel RET kinase inhibitor treatment. Methods: Wild-type and p.632_633del RET sequences were modeled using a lighter version of the AlphaFold2 (AF2) software. Functional studies were performed on transfected HEK 293 cells (pCMV6-Entry, pCMV6-RET, or pCMV6-RET(p.632_633del) treated with inhibitors for 24 hours and analyzed on luciferase assays. Results: Structural modeling revealed a paucity of disulfide bridge between Cys630-Cys634 in p.632_633del RET sequences, apparent in wild-type, while forming an intermolecular disulfide bridge between two Cys656. Proximity of juxtamembrane segments of each dimer may impede Tyr687 phosphorylation and stable conformation of intracellular RET that hosts selpercatinib. In vitro experiments confirmed a reduction in efficacy of selpercatinib upon p.632_633del RET compared with wild-type RET control. Conclusion: Clinical presentation together with structural modeling and functional studies suggests that p.632_633del RET results in poor response to selpercatinib.

Challenges and Strategies to Combat Resistance Mechanisms in Thyroid Cancer Therapeutics.

Background: BRAFV600E and N/H/K RAS mutations and oncogenic kinase fusions involving neurotrophin tyrosine receptor kinase (NTRK), RET, anaplastic lymphoma kinase (ALK), and ROS1 have been identified as actionable targets in thyroid cancer. These driver alterations lead to oncogene addiction, which has been successfully exploited through tyrosine kinase inhibitors. Acquired resistance may develop following an initial response requiring a therapeutic pivot to new therapies. Summary: Several pathways for development of acquired resistance have been identified. These encompass acquired on-target gene mutation impeding drug activity and upregulation of bypass kinase signaling pathways leading to tumor progression. Biopsy of resistant lesions (liquid or tissue) and subsequent molecular analysis can assist with new therapeutic strategies. Conclusions: Progression-free survival is curtailed by developing acquired resistance. To minimize this therapeutic liability, clinicians must be anticipatory in identifying the drivers and characterizing mechanisms of on-target resistance.

The length of FOXE1 polyalanine tract in congenital hypothyroidism: Evidence for a pathogenic role from familial, molecular and cohort studies.

Introduction: FOXE1 is required for thyroid function and its homozygous mutations cause a rare syndromic form of congenital hypothyroidism (CH). FOXE1 has a polymorphic polyalanine tract whose involvement in thyroid pathology is controversial. Starting from genetic studies in a CH family, we explored the functional role and involvement of FOXE1 variations in a large CH population. Methods: We applied NGS screening to a large CH family and a cohort of 1752 individuals and validated these results by in silico modeling and in vitro experiments. Results: A new heterozygous FOXE1 variant segregated with 14-Alanine tract homozygosity in 5 CH siblings with athyreosis. The p.L107V variant demonstrated to significantly reduce the FOXE1 transcriptional activity. The 14-Alanine-FOXE1 displayed altered subcellular localization and significantly impaired synergy with other transcription factors, when compared with the more common 16-Alanine-FOXE1. The CH group with thyroid dysgenesis was largely and significantly enriched with the 14-Alanine-FOXE1 homozygosity. Discussion: We provide new evidence that disentangle the pathophysiological role of FOXE1 polyalanine tract, thereby significantly broadening the perspective on the role of FOXE1 in the complex pathogenesis of CH. FOXE1 should be therefore added to the group of polyalanine disease-associated transcription factors.

Foxe1 Deletion in the Adult Mouse Is Associated With Increased Thyroidal Mast Cells and Hypothyroidism.

CONTEXT: Foxe1 is a key thyroid developmental transcription factor. Germline deletion results in athyreosis and congenital hypothyroidism. Some data suggest an ongoing role for maintaining thyroid differentiation. OBJECTIVE: We created a mouse model to directly examine the role of Foxe1 in the adult thyroid. METHODS: A model of tamoxifen-inducible Cre-mediated ubiquitous deletion of Foxe1 was generated in mice of C57BL/6J background (Foxe1flox/flox/Cre-TAM). Tamoxifen or vehicle was administered to Foxe1flox/flox/Cre mice aged 6-8 weeks. Blood was collected at 4, 12, and 20 weeks, and tissues after 12 or 20 weeks for molecular and histological analyses. Plasma total thyroxine (T4), triiodothyronine, and thyrotropin (TSH) were measured. Transcriptomics was performed using microarray or RNA-seq and validated by reverse transcription quantitative polymerase chain reaction. RESULTS: Foxe1 was decreased by approximately 80% in Foxe1flox/flox/Cre-TAM mice and confirmed by immunohistochemistry. Foxe1 deletion was associated with abnormal follicular architecture and smaller follicle size at 12 and 20 weeks. Plasma TSH was elevated in Foxe1flox/flox/Cre-TAM mice as early as 4 weeks and T4 was lower in pooled samples from 12 and 20 weeks. Foxe1 deletion was also associated with an increase in thyroidal mast cells. Transcriptomic analyses found decreased Tpo and Tg and upregulated mast cell markers Mcpt4 and Ctsg in Foxe1flox/flox/Cre-TAM mice. CONCLUSION: Foxe1 deletion in adult mice was associated with disruption in thyroid follicular architecture accompanied by biochemical hypothyroidism, confirming its role in maintenance of thyroid differentiation. An unanticipated finding was an increase in thyroidal mast cells. These data suggest a possible explanation for previous human genetic studies associating alleles in/near FOXE1 with hypothyroidism and/or autoimmune thyroiditis.

Contribution of a common variant in the promoter of the 1-α-hydroxylase gene (CYP27B1) to fracture risk in the elderly.

CYP27B1 encodes mitochondrial 1α-hydroxylase, which converts 25-hydroxyvitamin D to its active 1,25-dihydroxylated metabolite. We tested the hypothesis that common variants in the CYP27B1 promoter are associated with fracture risk. The study was designed as a population-based genetic association study, which involved 153 men and 596 women aged 65-101 years, who had been followed for 2.2 years (range 0.1-5.5) between 1999 and 2006. During the follow-up period, the incidence of fragility fractures was ascertained. Bone ultrasound attenuation (BUA) was measured in all individuals, as were serum 25-hydroxyvitamin D and PTH concentrations; 86% subjects had vitamin D insufficiency. Genotypes were determined for the -1260C>A (rs10877012) and +2838T>C (rs4646536) CYP27B1 polymorphisms. A reporter gene assay was used to assess functional expression of the -1260C>A CYP27B1 variants. The association between genotypes and fracture risk was analyzed by Cox's proportional hazards model. We found that genotypic distribution of CYP27B1 -1260 and CYP27B1 +2838 polymorphisms was consistent with the Hardy-Weinberg equilibrium law. The two polymorphisms were in high linkage disequilibrium, with D' = 0.96 and r² = 0.94. Each C allele of the CYP27B1 -1260 polymorphism was associated with increased risk of fracture (hazard ratio = 1.34, 95% CI 1.03-1.73), after adjustment for age, sex, number of falls, and BUA. In transient transfection studies, a reporter gene downstream of the -1260(A)-containing promoter was more highly expressed than that containing the C allele. These data suggest that a common but functional variation within the CYP27B1 promoter gene is associated with fracture risk in the elderly.

Translational Utility of Liquid Biopsies in Thyroid Cancer Management.

Liquid biopsies are a novel technique to assess for either circulating tumor cells (CTC) or circulating tumor DNA (ctDNA and microRNA (miRNA)) in peripheral blood samples of cancer patients. The diagnostic role of liquid biopsy in oncology has expanded in recent years, particularly in lung, colorectal and breast cancer. In thyroid cancer, the role of liquid biopsy in either diagnosis or prognosis is beginning to translate from the lab to the clinic. In this review, we describe the evolution of liquid biopsies in detecting CTC, ctDNA and miRNA in thyroid cancer patients, together with its limitations and future directions in clinical practice.

Key MicroRNA's and Their Targetome in Adrenocortical Cancer.

Adrenocortical Carcinoma (ACC) is a rare but aggressive malignancy with poor prognosis and limited response to available systemic therapies. Although complete surgical resection gives the best chance for long-term survival, ACC has a two-year recurrence rate of 50%, which poses a therapeutic challenge. High throughput analyses focused on characterizing the molecular signature of ACC have revealed specific micro-RNAs (miRNAs) that are associated with aggressive tumor phenotypes. MiRNAs are small non-coding RNA molecules that regulate gene expression by inhibiting mRNA translation or degrading mRNA transcripts and have been generally implicated in carcinogenesis. This review summarizes the current insights into dysregulated miRNAs in ACC tumorigenesis, their known functions, and specific targetomes. In addition, we explore the possibility of particular miRNAs to be exploited as clinical biomarkers in ACC and as potential therapeutics.

Congenital Hypoparathyroidism Associated With Elevated Circulating Nonfunctional Parathyroid Hormone Due to Novel PTH Mutation.

CONTEXT: Familial hypoparathyroidism has a heterogeneous presentation where patients usually have low parathyroid hormone (PTH) levels due to impaired production or secretion. This contrasts with pseudohypoparathyroidism, in which PTH resistance is usually associated with an elevated serum PTH. High levels of circulating PTH can also be due to bioinactive PTH, which is difficult to distinguish from pseudohypoparathyroidism on biochemical grounds. CASE DESCRIPTION: We report on 2 sisters from consanguineous parents who presented with tetany at birth and were diagnosed with congenital hypocalcemia. Serum PTH levels were normal for many years, but progressively increased in midadulthood to greater than 100x the upper limit of normal on multiple assays. Homozygosity mapping was performed on 1 sister that demonstrated loss of heterozygosity (LOH) around PTH. Sequencing revealed a previously unreported variant, c.94T>C, predicting a codon change of p.Ser32Pro that is biologically inactive. CONCLUSIONS: This case report shows a previously unreported unusual biochemical phenotype of a rising PTH in the context of a novel PTH mutation. This expands the evolving genotypes associated with hypoparathyroidism without established gene mutations.

ETS Factor ETV5 Activates the Mutant Telomerase Reverse Transcriptase Promoter in Thyroid Cancer.

Background: Co-occurrence of TERT (telomerase reverse transcriptase) promoter (TERTp) mutations with BRAF/RAS mutations is associated with significantly more aggressive thyroid cancer. TERTp mutations are hypothesized to generate de novo binding sites for ETS transcription factors, which are themselves activated by BRAF/RAS-stimulated MEK-ERK activity. To date, a detailed study of this mechanism has been limited to only a few cancer types, and we hypothesized that ETS factors involved in TERTp activation could vary between different cancers. Methodology: Here we sought to identify ETS factor(s) required for TERTp activation in thyroid cancer, using a combination of in silico analyses of TCGA data, and experimentation using in vitro thyroid cell models analyzed by quantitative reverse transcription-PCR, immunoprecipitation (IP), chromatin IP, and gene reporter assays. Results: We found that ETV5 was abundantly expressed in papillary thyroid cancers from the TCGA data set, and in thyroid cancer cell line models. Furthermore, ETV5 was found to preferentially bind to the -124 bp(T) TERTp allele and stimulate TERT transcription in thyroid cancer cells devoid of GA binding protein transcription factor (GABP) activity. We also found that ETV5 functionally cooperates with the transcription factor FOXE1 to further enhance TERTp activity, a mechanism that may at least partially explain why FOXE1 represents a significant genetic determinant of thyroid cancer risk. Conclusions: ETS factors that activate mutant TERTp vary between cancer types, and here we show for the first time that ETV5 demonstrates mutant allele-specific affinity for TERTp in thyroid cancer, a property that has previously only been attributable to GABP.

An InDel in Phospholipase-C-B-1 Is Linked with Euthyroid Multinodular Goiter.

BACKGROUND: Euthyroid multinodular goiter (MNG) is common, but little is known about the genetic variations conferring predisposition. Previously, a family with MNG of adolescent onset was reported in which some family members developed papillary thyroid carcinomas (PTC). METHODS: Genome-wide linkage analysis and next-generation sequencing were conducted to identify genetic variants that may confer disease predisposition. A multipoint nonparametric LOD score of 3.01 was obtained, covering 19 cM on chromosome 20p. Haplotype analysis reduced the region of interest to 10 cM. RESULTS: Analysis of copy number variation identified an intronic InDel (∼1000 bp) in the PLCB1 gene in all eight affected family members and carriers (an unaffected person who has inherited the genetic trait). This InDel is present in approximately 1% of "healthy" Caucasians. Next-generation sequencing of the region identified no additional disease-associated variant, suggesting a possible role of the InDel. Since PLCB1 contributes to thyrocyte growth regulation, the InDel was investigated in relevant Caucasian cohorts. It was detected in 0/70 PTC but 4/81 unrelated subjects with MNG (three females; age at thyroidectomy 27-59 years; no family history of MNG/PTC). The InDel frequency is significantly higher in MNG subjects compared to controls (χ2 = 5.076; p = 0.024. PLCB1 transcript levels were significantly higher in thyroids with the InDel than without (p < 0.02). CONCLUSIONS: The intronic PLCB1 InDel is the first variant found in familial multiple papilloid adenomata-type MNG and in a subset of patients with sporadic MNG. It may function through overexpression, and increased PLC activity has been reported in thyroid neoplasms. The potential role of the deletion as a biomarker to identify MNG patients more likely to progress to PTC merits exploration.

NRASQ61R Mutation-specific Immunohistochemistry Also Identifies the HRASQ61R Mutation in Medullary Thyroid Cancer and May Have a Role in Triaging Genetic Testing for MEN2.

A quarter of patients with medullary thyroid carcinoma (MTC) have germline mutations in the RET proto-oncogene indicating MEN2. Therefore genetic testing is recommended for all patients presenting with MTC. Approximately 40% of MTCs have somatic RET mutations. Somatic mutations in the RAS genes are the next most common driver mutations and appear to be mutually exclusive with germline RET mutation. The single most common somatic RAS mutation is HRASQ61R (c.182A>G), reported in 4.6% to 11% of all MTCs. Mutation-specific immunohistochemistry (IHC) initially developed to identify the NRASQ61R mutation in melanoma (clone SP174) has proven highly sensitive and specific. Because the amino acid sequences for the HRAS and NRAS proteins at codon 61 are identical, we postulated that SP174 IHC would also identify the somatic HRASQ61R mutation. IHC with SP174 was performed on a tissue microarray of 68 patients with MTC including 13 (22.8%) with molecularly confirmed MEN2. Seven (10.3%) MTCs demonstrated positive staining. Six of these patients had already undergone germline RET mutation testing as part of clinical care and were all confirmed to be wild type, excluding the diagnosis of MEN2. All SP174 immunohistochemically positive MTCs were proven to have HRASQ61R mutation (and lack KRASQ61R and NRASQ61R) by Sanger sequencing. All MEN2 patients showed negative staining. We conclude that IHC with SP174 is highly specific for the HRASQ61R mutation in MTC. Because current data suggest that this mutation is mutually exclusive with germline RET mutation, IHC may also have a role in triaging formal genetic testing for MEN2.

Altered tear composition in smokers and patients with graves ophthalmopathy.

OBJECTIVES: To analyze, compare, and contrast tear composition in healthy nonsmokers, smokers who were otherwise healthy, and patients with Graves disease (GD) accompanied by Graves ophthalmopathy (GO) of varying severity. METHODS: Reflex tears were collected using Schirmer strips from 37 healthy nonsmokers, 33 otherwise healthy smokers, 51 patients with GD, and 85 patients with GO. Thyrotropin receptor-stimulating activity and serum thyroid-stimulating antibodies were measured. Pooled tear samples from healthy nonsmokers, healthy smokers, and patients with GO were separated by means of electrophoresis. Proteins expressed in healthy smokers, patients with GO, and healthy nonsmokers were separated by means of electrophoresis and analyzed by mass spectrometry. RESULTS: Based on the 97th percentile of findings from healthy nonsmokers, specific thyrotropin receptor-stimulating activity was detected in 25% of the tear samples from healthy smokers, 32% of those from patients with GD, and 41% of those from patients with GO. Clinical activity scores correlated with serum thyroid-stimulating antibody levels but not tear thyrotropin receptor-stimulating activity. Electrophoresis revealed additional proteins of 30 to 41 kDa in the tear samples from patients with GO and healthy smokers compared with samples from healthy nonsmokers. These proteins were identified as zinc-alpha2-glycoprotein and lactoferrin but have no thyrotropin receptor-stimulating activity. CONCLUSIONS: We demonstrate similar changes in tear composition in patients with GO and healthy smokers compared with healthy nonsmokers. Expression of zinc-alpha2-glycoprotein and lactoferrin is increased and their molecular weights are modified, suggesting degradation and/or changes during glycosylation, which may affect the bioactivities of zinc-alpha2-glycoprotein and lactoferrin. Clinical Relevance Smoking, which is a significant risk factor for the development of GO, modifies tear composition.

DNA Methylation Analysis: Choosing the Right Method.

In the burgeoning field of epigenetics, there are several methods available to determine the methylation status of DNA samples. However, choosing the method that is best suited to answering a particular biological question still proves to be a difficult task. This review aims to provide biologists, particularly those new to the field of epigenetics, with a simple algorithm to help guide them in the selection of the most appropriate assay to meet their research needs. First of all, we have separated all methods into two categories: those that are used for: (1) the discovery of unknown epigenetic changes; and (2) the assessment of DNA methylation within particular regulatory regions/genes of interest. The techniques are then scrutinized and ranked according to their robustness, high throughput capabilities and cost. This review includes the majority of methods available to date, but with a particular focus on commercially available kits or other simple and straightforward solutions that have proven to be useful.

Thyroid transcription factor FOXE1 interacts with ETS factor ELK1 to co-regulate TERT.

BACKGROUND: Although FOXE1 was initially recognized for its role in thyroid organogenesis, more recently a strong association has been identified between the FOXE1 locus and thyroid cancer. The role of FOXE1 in adult thyroid, and in particular regarding cancer risk, has not been well established. We hypothesised that discovering key FOXE1 transcriptional partners would in turn identify regulatory pathways relevant to its role in oncogenesis. RESULTS: In a transcription factor-binding array, ELK1 was identified to bind FOXE1. We confirmed this physical association in heterologously transfected cells by IP and mammalian two-hybrid assays. In thyroid tissue, endogenous FOXE1 was shown to bind ELK1, and using ChIP assays these factors bound thyroid-relevant gene promoters TPO and TERT in close proximity to each other. Using a combination of electromobility shift assays, TERT promoter assays and siRNA-silencing, we found that FOXE1 positively regulated TERT expression in a manner dependent upon its association with ELK1. Treating heterologously transfected thyroid cells with MEK inhibitor U0126 inhibited FOXE1-ELK1 interaction, and reduced TERT and TPO promoter activity. METHODOLOGY: We investigated FOXE1 interactions within in vitro thyroid cell models and human thyroid tissue using a combination of immunoprecipitation (IP), chromatin IP (ChIP) and gene reporter assays. CONCLUSIONS: FOXE1 interacts with ELK1 on thyroid relevant gene promoters, establishing a new regulatory pathway for its role in adult thyroid function. Co-regulation of TERT suggests a mechanism by which allelic variants in/near FOXE1 are associated with thyroid cancer risk.

Somatic Mutations of FOXE1 in Papillary Thyroid Cancer.

BACKGROUND: Population-based studies have demonstrated an association of single nucleotide polymorphisms close to the thyroid transcription factor forkhead box E1 (FOXE1) gene with thyroid cancer. The dysregulation of forkhead proteins is increasingly recognized to play a role in the development and progression of cancer. The objective of the study was to seek to identify novel mutations in FOXE1 in papillary thyroid cancer (PTC) and to assess the effect of these mutations on protein expression and transcriptional function on FOXE1 responsive promoters. METHODS: The study was conducted at two tertiary referral hospitals. The coding region of FOXE1 was sequenced in tissue-derived DNA or RNA from 120 patients with PTC and 110 patients with multinodular goiter (MNG). In vitro studies were performed to examine the protein expression and transcriptional function of FOXE1 mutants. A molecular model of the forkhead domain (FHD) of FOXE1 was generated using the SWISS-MODEL online server with the three-dimensional structure of FOXD3 as a template. RESULTS: Three somatic missense mutations were detected in PTC resulting in the amino acid substitutions P54Q, K95Q, and L112F. One additional mutation was detected in a MNG (G140R). In vitro studies demonstrated marked impairment in transcriptional activation by all four FOXE1 mutants, which was not explained by differences in protein expression. Molecular modeling localized three of the mutations to highly conserved regions of the FHD. CONCLUSIONS: We have identified novel somatic mutations of FOXE1 in PTC. Mutational inactivation of FOXE1 is an uncommon event in thyroid tumors but may contribute to thyroid carcinogenesis and dedifferentiation in concert with other oncogenic drivers.