Thyroid Cells Research Articles

Pathomorphological characteristics of heat stress in the experiment.

The purpose of this research was to explore some morphological, physiological, and biochemical changes in female and fetal Wistar rats under heat stress. The experiment involved 30 animals, including two experimental groups (pregnant and nonpregnant females) kept under heat stress at 32°C and one control group consisting of healthy individuals kept in standard vivarium conditions. After dissection, fixation, dehydration, and primary processing, tissue samples were embedded in a mixture of paraffin and lanolin to obtain material for sections. Sections were made using a freezing and angular microtome and stained with hematoxylin and fuchsine solutions. Changes in morphology were assessed by microscopy using a Leitz DIAPLAN system. As a result of heat stress, an increase in linear cell size, capillary network area, and adrenal mass was observed; adipocytes lost lipid vacuoles; prismatic thyroid cells were replaced by flat cells; hypothyroidism; an increase in the number of osteocyte lacunae; and increased osteoclast activity in bone tissue; interstitial and intracellular oedema and caryopycnosis of ventricular cardiomyocytes; reduction in the diameter of skeletal muscle fibers and replacement of tissue with collagen fibers; water loss in the structure of myofibrils; destructive local changes, hyperchromatosis and caryopycnosis of the hippocampus. The data obtained allows predicting the possible consequences of prolonged overheating of tissues of other vertebrates and the human body.

DsRNAi-mediated silencing of PIAS2beta specifically kills anaplastic carcinomas by mitotic catastrophe

The E3 SUMO ligase PIAS2 is expressed at high levels in differentiated papillary thyroid carcinomas but at low levels in anaplastic thyroid carcinomas (ATC), an undifferentiated cancer with high mortality. We show here that depletion of the PIAS2 beta isoform with a transcribed double-stranded RNA–directed RNA interference (PIAS2b-dsRNAi) specifically inhibits growth of ATC cell lines and patient primary cultures in vitro and of orthotopic patient-derived xenografts (oPDX) in vivo. Critically, PIAS2b-dsRNAi does not affect growth of normal or non-anaplastic thyroid tumor cultures (differentiated carcinoma, benign lesions) or cell lines. PIAS2b-dsRNAi also has an anti-cancer effect on other anaplastic human cancers (pancreas, lung, and gastric). Mechanistically, PIAS2b is required for proper mitotic spindle and centrosome assembly, and it is a dosage-sensitive protein in ATC. PIAS2b depletion promotes mitotic catastrophe at prophase. High-throughput proteomics reveals the proteasome (PSMC5) and spindle cytoskeleton (TUBB3) to be direct targets of PIAS2b SUMOylation at mitotic initiation. These results identify PIAS2b-dsRNAi as a promising therapy for ATC and other aggressive anaplastic carcinomas.

A rare clinical case of malignant ectopic retrosternal goiter of a large size in a patient with aberrant right subclavian artery

Background. Ectopic goiter accounts for only 0.2 % to 1 % of all mediastinal goiters. The aberrant right subclavian artery (arteria lusoria) is an extremely rare anomaly of the major vessel, with a prevalence not exceeding 0.3 % in the general population. This paper presents the first case of malignant ectopic retrosternal goiter of a large size in combination with the aberrant right subclavian artery. Clinical case description. A 61-year-old male presented to the thoracic surgery department of P. Herzen Moscow oncology Research institute with a large posterior mediastinal mass measuring 108×106×79 mm and adjacent to the right lobe of the thyroid gland. The tumor displaced the trachea and esophagus. Open biopsy through a thoracotomy incision revealed thyroid gland cells without signs of atypia. A detailed examination revealed mediastinal vascular anomaly – retrotracheal position of the right subclavian artery. a complete longitudinal sternotomy, complemented by a right-sided thoracotomy at the 3rd intercostal space, served as the surgical approach. The operation lasted 320 minutes, with a blood loss of 3500 ml. The postoperative period was uneventful. The morphological examination of the surgical specimen revealed nodular hyperplasia of the ectopic thyroid tissue with the growth of well-differentiated follicular carcinoma without metastatic involvement of removed lymph nodes. Considering the identified follicular carcinoma, total thyroidectomy was performed. Conclusion. In surgical planning, it is crucial to assess the location of major mediastinal vessels and their relationship with the goiter. Therefore, all patients with mediastinal lesions should undergo preoperative contrast-enhanced ct. Retrosternal goiter predisposes to a higher rate of malignancy compared to cervical goiter. If the lower edge of the goiter is at the level of or below the aortic arch, cervical access should be supplemented with sternotomy. In cases of a massive thoracic component, additional thoracotomy is justified.

Identification of Iodotyrosines as Novel Substrates for the Thyroid Hormone Transporter MCT8.

Background: Monocarboxylate transporter 8 (MCT8) is the most specific thyroid hormone transporter identified to date, deficiency of which has been associated with severe intellectual and motor disability and abnormal serum thyroid function tests. However, it is presently unknown if MCT8, similar to other thyroid hormone transporters, also accepts additional substrates, and if disruption of their transport may contribute to the observed phenotype. Methods: In this study, we aimed to identify such substrates by applying liquid chromatography-mass spectrometry-based metabolome analysis in lysates of control and MCT8-overexpressing Xenopus oocytes. A subset of identified candidate substrates were validated by direct transport studies in transiently transfected COS-1 cells and human fibroblasts, which endogenously express MCT8. Moreover, transport characteristics were determined, including transport saturation and cis-inhibition potency of thyroid hormone transport. Results: Metabolome analysis identified 21 m/z ratios, corresponding to 87 candidate metabolites, with a 2.0-times differential abundance in MCT8-injected oocytes compared with controls. These metabolites included 3,5-diiodotyrosine (DIT) and several amino acids, including glutamate and glutamine. In accordance, MCT8-expressing COS-1 cells had 2.2-times lower intracellular accumulation of [125I]-DIT compared with control cells. This effect was largely blocked in the presence of 3,3',5-triiodothyronine (T3) (IC50: 2.5 ± 1.5 µM) or thyroxine (T4) (IC50: 5.8 ± 1.3 µM). Conversely, increasing concentrations of DIT enhanced the accumulation of T3 and T4. The MCT8-specific inhibitor silychristin increased the intracellular accumulation of DIT in human fibroblasts. COS-1 cells expressing MCT8 also exhibited a 50% reduction in intracellular accumulation of [125I]-3-monoiodotyrosine (MIT). In contrast, COS-1 cells expressing MCT8 did not alter the intracellular accumulation of [3H]-glutamate or [3H]-glutamine. However, studies in human fibroblasts showed a 1.5-1.9 times higher glutamate uptake in control fibroblasts compared with fibroblasts derived from patients with MCT8 deficiency, which was not affected in the presence of silychristin. Conclusions: Taken together, our results suggest that the iodotyrosines DIT and MIT can be exported by MCT8. MIT and DIT interfere with MCT8-mediated transport of thyroid hormone in vitro and vice versa. Future studies should elucidate if MCT8, being highly expressed in thyroidal follicular cells, also transports iodotyrosines invivo.

Do PFCAs drive the establishment of thyroid cancer microenvironment? Effects of C6O4, PFOA and PFHxA exposure in two models of human thyroid cells in primary culture

BackgroundExposure to environmental pollutants is suspected to be one of the potential causes accounting for the increase in thyroid cancer (TC) incidence worldwide. Among the ubiquitous pollutants, per-polyfluoroalkyl substances (PFASs), were demonstrated to exert thyroid disrupting effects. Perfluoroalkyl carboxylates (PFCAs) represent a subgroup of PFAS and include perfluoro carboxylic acids (PFOA and PFHxA) and perfluoropolyether carboxylic acid (C6O4).The potential relationship between exposure to PFCAs and TC was not yet fully elucidated.This in vitro study investigated whether certain PFCAs (C6O4, PFOA, and PFHxA) can influence the composition of TC microenvironment. MethodsTwo models of normal thyroid cells in primary cultures: Adherent (A-NHT) and Spheroids (S-NHT) were employed. A-NHT and S-NHT were exposed to C6O4, PFOA or PFHxA (0; 0.01; 0.1, 1; 10; 100; 1000 ng/mL) to assess viability (WST-1 and AV/PI assay), evaluate spherification index (SI) and volume specifically in S-NHT. CXCL8 and CCL2 (mRNA and protein), and EMT-related genes were assessed in both models after exposure to PFCAs. ResultsPFHxA reduced the viability of both A-NHT and S-NHT. None of the PFCAs interfered with the volume or spherification process in S-NHT. CXCL8 and CCL2 mRNA and protein levels were differently up-regulated by each PFCAs, being PFOA and PFHxA the stronger inducers. Moreover, among the tested PFCAs, PFHxA induced a more consistent increase in the mRNA levels of EMT-related genes. Conclusions: This is the first evaluation of the effects of exposure to PFCAs on factors potentially involved in establishing the TC microenvironment. PFHxA modulated the TC microenvironment at three levels: cell viability, pro-tumorigenic chemokines, and EMT-genes. The results provide further evidence of the pro-tumorigenic effect of PFOA. On the other hand, a marginal effect was observed for C6O4 on pro-tumorigenic chemokines.

Investigation of Dextran-Coated Magnetic Nanoparticles Encapsulated by medication and modified with Folate for Targeted drug delivery: Invito and docking studies

In this study, we synthesized dextran (DEX)-coated iron oxide (Fe3O4) and encapsulated it by the anticancer drug cisplatin (CP) . This nano-composite functionalized with folic acid (FA) molecules for targeted delivery. These nanoparticles (NPs) were then used as nanocarriers for the targeted administration of CP for treating thyroid tumor cancer. The Fe3O4 NPs were synthesized using a chemical technique. The development of the Fe3O4@DEX-CP-FA nanosystem was achieved using a reverse microemulsion technique. The morphological features of the NPs were characterized using several methods, including dynamic light scattering, atomic force microscopy, vibrating sample magnetometry, high-resolution transmission electron microscopy, and field emission scanning electron microscopy. The docking study has verified that nanoparticles have anti-proliferative effects in thyroid cancer by attaching strongly to molecular targets. The MTT test evaluated the effects of a curative Fe3O4@DEX-CP-FA effect on cancerous and non-cancerous cell lines. The results obtained from the formulation of Fe3O4@DEX-CP-FA revealed the presence of spherical particles with an average size of 107.3 nm, a zeta potential of -28.7 mV, and a polydispersity value of 0.055 mV. Depending on the dose, the nanosystem exhibited cytotoxic effects on thyroid tumor (TT) and normal human thyroid (NHT) cell lines. The study findings indicate that Fe3O4@DEX-CP-FA demonstrated sustained antitumor efficacy while not revealing a harmful impact on normal cells.

An Expedition on Synthetic Methodology of FDA-approved Anticancer Drugs (2018-2021).

New drugs being established in the market every year produce specified structures for selective biological targeting. With medicinal insights into molecular recognition, these begot molecules open new rooms for designing potential new drug molecules. In this review, we report the compilation and analysis of a total of 56 drugs including 33 organic small molecules (Mobocertinib, Infigratinib, Sotorasib, Trilaciclib, Umbralisib, Tepotinib, Relugolix, Pralsetinib, Decitabine, Ripretinib, Selpercatinib, Capmatinib, Pemigatinib, Tucatinib, Selumetinib, Tazemetostat, Avapritinib, Zanubrutinib, Entrectinib, Pexidartinib, Darolutamide, Selinexor, Alpelisib, Erdafitinib, Gilteritinib, Larotrectinib, Glasdegib, Lorlatinib, Talazoparib, Dacomitinib, Duvelisib, Ivosidenib, Apalutamide), 6 metal complexes (Edotreotide Gallium Ga-68, fluoroestradiol F-18, Cu 64 dotatate, Gallium 68 PSMA-11, Piflufolastat F-18, 177Lu (lutetium)), 16 macromolecules as monoclonal antibody conjugates (Brentuximabvedotin, Amivantamab-vmjw, Loncastuximabtesirine, Dostarlimab, Margetuximab, Naxitamab, Belantamabmafodotin, Tafasitamab, Inebilizumab, SacituzumabGovitecan, Isatuximab, Trastuzumab, Enfortumabvedotin, Polatuzumab, Cemiplimab, Mogamulizumab) and 1 peptide enzyme (Erwiniachrysanthemi-derived asparaginase) approved by the U.S. FDA between 2018 to 2021. These drugs act as anticancer agents against various cancer types, especially non-small cell lung, lymphoma, breast, prostate, multiple myeloma, neuroendocrine tumor, cervical, bladder, cholangiocarcinoma, myeloid leukemia, gastrointestinal, neuroblastoma, thyroid, epithelioid and cutaneous squamous cell carcinoma. The review comprises the key structural features, approval times, target selectivity, mechanisms of action, therapeutic indication, formulations, and possible synthetic approaches of these approved drugs. These crucial details will benefit the scientific community for futuristic new developments in this arena.

Biological functions of LncRNA SNHG14 in the development of thyroid cancer cells via targeting miR-206.

This study aimed to determine lncRNA SNHG14 and miR-206 in Thyroid Cancer ( TC) and to explore the related mechanisms. Sixty-four samples of thyroid tissue were collected from patients with TC. TC cell lines and a normal human thyroid cell line (HTori-3) were bought. lncRNA SNHG14-siRNA (si-lncRNA SNHG14), lncRNA SNHG14-shRNA (sh-lncRNA SNHG14), blank plasmid (siRNA-NC), miR-206-inhibitor, miR-206-mimics were transfected into BHT101 and Ocut-2C cells. qRT-PCR quantified the expression of lncRNA SNHG14 and miR-206, and the expression of vimentin, Snail, N-cadherin, Slug, E-cadherin and ZO-1 proteins were identified via WB. MTT assay, flow cytometry, and Transwell were employed to determine cellular proliferation, apoptosis, and invasion, separately. The high expression of lncRNA SNHG14 and low expression of miR-206 were exhibited in patients with TC. lncRNA SNHG14 and miR-206 were related to lymph node metastases, TNM staging, as well as differentiation of TC. Silencing lncRNA SNHG14 and over-expressing miR-206 inhibited cell EMT, proliferation, and invasion, but accelerated apoptosis. WB demonstrated that silencing lncRNA SNHG14 and over-expressing miR-206 suppressed the expression of Akt, p-ERK1/2, p-p38, p-4EBP1, p-Akt, PI3K, vimentin, Snail, N-cadherin, and Slugn, as well as up-regulated the expression of E-cadherin and ZO-1. Rescue experiment showed that after BHT101 and Ocut-2C cells were transfected with either sh-lncRNA SNHG14+miR-206-mimics or si-lncRNA SNHG14+miR-206-inhibitor, the cellular proliferative, invasive, and apoptotic activities weren't different from those transfected with siRNA-NC. Suppression of lncRNA SNHG14 up-regulates miR-206 and affects EMT, as well as proliferative, invasive, and apoptotic activities of cells, which may become an underlying treatment target for TC.

TAF1 is needed for the proliferation and maturation of thyroid follicle cells via Notch signaling.

Thyroid dysgenesis (TD) is the common pathogenic mechanism of congenital hypothyroidism (CH). In addition, known pathogenic genes are limited to those that are directly involved in thyroid development. To identify additional candidate pathogenetic genes, we performed forward genetic screening for TD in zebrafish, followed by positional cloning. The candidate gene was confirmed in vitro using the Nthy-ori 3.1 cell line and in vivo using a zebrafish model organism. We obtained a novel zebrafish line with thyroid dysgenesis and identified the candidate pathogenetic gene taf1 by positional cloning. Further molecular studies revealed that taf1 was needed for the proliferation of thyroid follicular cells by binding to the NOTCH1 promoter region. Knockdown of TAF1 impaired the proliferation and maturation of thyroid cells, thereby leading to thyroid dysplasia. This study showed that TAF1 promoted Notch signaling and that this association played a pivotal role in thyroid development.

Diagnostic, Theranostic and Prognostic Value of Thyroglobulin in Thyroid Cancer.

Thyroglobulin (Tg) is an iodinated glycoprotein, which is normally stored in the follicular colloid of the thyroid, being a substrate for thyroid hormone production. Since it is produced by well-differentiated thyroid cells, it is considered a reliable tumor marker for patients with differentiated thyroid carcinoma (DTC) during their follow-up after total thyroidectomy and radioiodine ablation. It is used to monitor residual disease and to detect recurrent disease. After total thyroid ablation, unstimulated highly sensitive Tg measurements are sufficiently accurate to avoid exogenous or endogenous thyrotropin (TSH) stimulation and provide accurate diagnostic and prognostic information in the great majority of DTC patients. Adopting sophisticated statistical analysis, i.e., decision tree models, the use of Tg before radioiodine theranostic administration was demonstrated to be useful in refining conventional, pathology-based risk stratification and providing personalized adjuvant or therapeutic radioiodine administrations. The follow-up of DTC patients aims to promptly identify patients with residual or recurrent disease following primary treatment. Our review paper covers the diagnostic, theranostic and prognostic value of thyroglobulin in DTC patients.

Near-infrared fluorescent probe based on carboxylesterase 2 and viscosity cascade response for early diagnosis of thyroid cancer

Carboxylesterase 2 (CES2) plays a critical role in the metabolism of ester- and amide-containing drugs and thus has an impact on drug efficacy and toxicity. This study introduces QVJ-CES2, a near-infrared fluorescent probe designed to enhance thyroid cancer diagnosis through CES2 enzyme activity and distinct cellular viscosity of thyroid cells. The probe's dual-response mechanism significantly amplified fluorescence in high-viscosity environments, thus had high sensitivity. Our findings revealed that QVJ-CES2 could detect CES2 activity with a limit of detection as low as 1.9 ng/mL. In vitro experiments confirmed the rapid and selective response of QVJ-CES2 to CES2 and the ability to distinguish cancer cells from their normal cells with high specificity. Most importantly, the fluorescence intensity difference between cancerous and normal cells after incubation with the probe was as high as 7-fold, attributable to the synergistic enzyme and viscosity cascade response. Further, in vivo studies demonstrated that the probe was effective in accurately identifying thyroid cancer tissues, showing that it is a promising tool for early cancer detection and precise surgical interventions.

Concomitant lithium increases radioiodine uptake and absorbed doses per administered activity in graves' disease: comparison of conventional versus lithium-augmented radioiodine therapy.

BackgroundLithium inhibits iodine and thyroid hormone release from thyroid cells, possibly increasing radioiodine retention and anti-hyperthyroid efficacy when given adjunctively to radioiodine therapy (RAI) of Graves’ disease (GD). However, the literature contains limited dosimetric data regarding the influence of concomitant lithium in this setting.MethodsWe retrospectively compared dosimetric variables in patients undergoing RAI with/without adjunctive lithium (n = 52 each). We assessed two low-dose, short-duration oral lithium carbonate regimens, 450 mg/d (n = 22) or 900 mg/d (n = 30), for a mean of 4.7 ± 1.4 d starting upon RAI administration. Patients underwent diagnostic testing to measure thyroidal radioiodine uptake (RAIU) 24 h ± 2 h after ingesting up to 5 MBq radioiodine, receiving individualized RAI activities 24 h later. Using ≥3 RAIU daily measurements starting 24 h post-RAI, researchers were able to determine the effective radioiodine half-life and absorbed dose to the thyroid; we also calculated the absorbed dose per administered activity concentration within that organ. Rates of GD cure, defined as reaching euthyroidism or hypothyroidism post-RAI, were evaluated in patients with ~6 months or longer post-RAI follow-up.ResultsThe lithium dosage subgroups had similar dosimetric values and thus are considered together. Lithium patients and controls had similar average “diagnostic” RAIU (51.1% ± 15.7% vs. 50.6% ± 13.8%, p = 0.820), but the former had significantly higher RAIU post-RAI (56.3% ± 13.5% vs. 49.1% ± 13.5%, p = 0.002), reflecting significantly greater change in the former (+16.2% ± 30.4% vs. -1.8% ± 16.1%, p = 0.001). Radioiodine effective half-life was non-significantly longer in lithium patients (5.43 ± 1.50 d vs. 5.08 ± 1.16 d, p = 0.192). The mean RAI administered activity was 27% less in lithium patients (677 ± 294 MBq vs. 930 ± 433 MBq, p = 0.001), but GD cure rates were similar (83% [39/47] vs. 82% [33/40], p = 0.954), possibly due to the significantly higher thyroid dose in the lithium patients, especially in thyroid gland with a volume ≤ 20 mL (1.04 ± 0.44 Gy/MBq vs. 0.76 ± 0.30 Gy/MBq, p = 0.020). Day 3 serum lithium concentrations were low (450 mg/d: 0.26 ± 0.12 mmol/L, 900 mg/d: 0.50 ± 0.18 mmol/L); no lithium toxicity was noted.ConclusionLithium augmentation may increase the RAIU and thyroid absorbed dose, permitting potentially decreased RAI activities without sacrificing efficacy. Our observations should be confirmed in a prospective, randomized trial.

Correlation between Interleukin-23, Autoantibodies and Thyroid Profile in a Sample of Iraqi Patients with Hashimoto’s Thyroiditis

Hashimoto’s thyroiditis (HT) is the most common autoimmune condition characterized by hypothyroidism and thyroid cell death by leukocytes and antibody-mediated immunological mechanisms. The current paper set out to assess a number of inflammatory and metabolic potential indicators of Hashimoto’s thyroiditis. Fifty-one patients with Hashimoto’s thyroiditis took part in the current investigation. Ages ranged from 20 to 75 for them. Enzyme-linked immunosorbent tests were used to quantify the anti-thyroperoxidase antibody (anti-TPO Ab), anti-thyroglobulin antibody (anti-Tg Ab), T4, T3, and TSH at the beginning and conclusion of the trial. The result showed a significant relationship between the gender according to HT patient’s and healthy groups which showed that female effected more than male and there was no effected according to the age between these groups, In HT patients with greater TSH concentrations compared to healthy individuals, serum FT3 and FT4 concentrations were lower while IL-23 concentrations were higher. TSH levels were positively correlated with IL-23 levels. As a result, IL-23 can be useful for detecting disease activity in HT patients. However, more thorough, extensive, longitudinal investigations are required to evaluate whether IL-23 can be used in therapeutic settings. Our results showed that thyroid profile, serum IL-23, and autoantibodies were strong indicators of Hashimoto’s thyroiditis. To better understand these correlations and underlying pathogenic mechanisms, additional research is necessary.

Identification of P21 (CDKN1A) Activated Kinase 4 as a Susceptibility Gene for Familial Non-Medullary Thyroid Carcinoma.

Background: Familial non-medullary thyroid carcinoma (FNMTC) is a genetically predisposed disease with unclear genetic mechanisms. This makes research on susceptibility genes important for the diagnosis and treatment options. Methods: This study included a five-member family affected by papillary thyroid carcinoma. The candidate genes were identified through whole-exome sequencing and Sanger sequencing in family members, other FNMTC patients, and sporadic non-medullary thyroid carcinoma patients. The pathogenicity of the mutation was predicted using in silico tools. Cell phenotype experiments in vitro and models of lung distant metastasis in vivo were conducted to confirm the characteristics of the mutation. Transcriptome sequencing and mechanistic validation were employed to compare the disparities between PAK4 wild-type (WT) and PAK4 mutant (MUT) cell lines. Results: This mutation alters the protein structure, potentially increasing instability by affecting hydrophobicity, intra-molecular hydrogen bonding, and phosphorylation sites. It specifically promotes phosphorylated PAK4 nuclear translocation and expression in thyroid tissue and cell lines. Compared with the WT cells line, PAK4 I417T demonstrates enhanced proliferation, invasiveness, accelerated cell division, and inhibition of cell apoptosis in vitro. In addition, it exhibits a significant propensity for metastasis in vivo. It activates tumor necrosis factor signaling through increased phosphorylation of PAK4, JNK, NFκB, and c-Jun, unlike the WT that activates it via the PAK4-NFκ-MMP9 axis. In addition, PAK4 MUT protein interacts with matrix metalloproteinase (MMP)3 and regulates MMP3 promoter activity, which is not observed in the WT. Conclusions: Our study identified PAK4: c.T1250C: p.I417T as a potential susceptibility gene for FNMTC. The study concludes that the mutant form of PAK4 exhibits oncogenic function, suggesting its potential as a novel diagnostic molecular marker for FNMTC.

Abstract 3591: Upregulation of CXCL8 is an early step in the oncogenic transition of human thyroid cells by BRAFV600E

Abstract Introduction: BRAFV600E is one of the most common oncogenic mutations in human thyroid cancers, especially in papillary thyroid carcinoma (PTC). However, the early molecular changes caused by this mutation have not been studied previously in human thyrocytes in vitro. Methods: Primary cultures of cells derived from noncancerous thyroid tissue of patients (at least n=3) were infected with BRAFV600E adenovirus to mimic the mutated condition. Changes induced by the infection were measured at early times (<24h). Transcriptome analysis was performed on infected thyrocytes (n=6) to determine the highest differentially expressed genes between the control and infected groups, which were then verified at the gene and protein levels. RNAi downregulation experiments were performed to determine the cross-regulatory function of the genes. Results: The induction of BRAFV600E mutation, confirmed at the protein level, increased proliferation of the cells, and morphological changes were observed at approximately 24h post-infection. The decreased expression of thyroid-specific genes, viz. TSHR, TG, and TPO were observed after >72h post-introduction of BRAFV600E mutation. Transcriptome and protein expression analysis of BRAFV600E-positive thyrocytes showed the overexpression of chemokines CXCL8 and CXCL12, and upregulation of endothelial cell-specific molecule 1 (ESM1). These molecules are known to be associated with increased neoangiogenesis and proliferation. Functional studies showed that siRNA knockdown of CXCL8 decreased the expressions of CXCL12 and ESM1, whereas knockdown of either CXCL12 or ESM1 did not decrease the expression of CXCL8. Conclusion: Increased proliferation, de-differentiation, upregulation of chemokines, and endothelial-specific factor are effects associated with BRAFV600E-driven thyroid carcinogenesis. CXCL8 appears to play an important early regulatory role in this process. Citation Format: Rhitajit Sarkar, Priyanka Bolel, Joanna Klubo-Gwiezdzinska, Marvin C. Gershengorn. Upregulation of CXCL8 is an early step in the oncogenic transition of human thyroid cells by BRAFV600E [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3591.

Abstract 4633: Differentiation driver gene HOXD4 as a potential prognostic indicator and therapeutic target in anaplastic thyroid cancer

Abstract Anaplastic thyroid cancer (ATC) is always diagnosed as stage IV with a 5 year survival rate of less than 3% due to its aggressive metastatic phenotype. ATC lacks differentiation and loses its thyroid-like functions making it a difficult cancer to treat due to its lack of targetable genetic lesions and unresponsiveness to conventional treatments. Therefore, we investigated genes that had aberrant expression in ATC in relation to differentiation. Using NCBI Gene expression omnibus (GSE33630, GSE85457), which provides publicly available transcriptomic datasets, and bioinformatics software, GEO2R, we found that Homeobox D4 (HOXD4) has 12.5FC higher expression in ATC vs. normal tissue (p=0.000552) and 4.36FC higher in ATC vs. papillary thyroid cancer (PTC) in human patients (p=6.69e-08). HOXD4 is a transcription factor that plays a crucial role in cell differentiation and has been associated with a worse prognosis in cancers such as ovarian serous carcinoma. As loss of differentiation is essential to ATC’s refractory nature and therapeutic resistance, we are exploring modulation of differentiation by all-trans retinoic acid (ATRA). ATRA was used to induce differentiation in ATC cell line T238 therefore abrogating the aggressive phenotype of this cancer. T238 treated with ATRA had a 26% decrease in proliferation at 24 hours, 46% at 48 hours, and 87% at 72 hours measured using a trypan blue exclusion assay. T238 treated with ATRA also had a 44% decrease in migratory capacity at 24 hours and 24% at 48 hours measured by a cell scratch wound assay. Immortalized normal thyroid cell line NTHY-ori-3-1 did not have a significant decrease in either proliferation or migratory capacity when treated with ATRA. T238 cells treated with ATRA for 24 hours had a 30% decrease in HOXD4 protein levels. Lastly, immunohistochemistry staining of HOXD4 in human patient tissue samples confirmed the over-expression of HOXD4 in ATC (19.5% staining) vs. normal tissue (2.5%) and PTC (7.5%). Overall, we hypothesize that the aberrant expression of HOXD4 in ATC plays a crucial role in the aggressive metastatic phenotype and therefore differentiation agents such as ATRA can target this biomarker to modulate the ATC phenotype. Therefore, HOXD4 can be used as a potential prognostic indicator for early detection of ATC. Future studies will involve CRISPRi based knockdown of HOXD4 in vitro and validation of these results in animal in vivo models. Citation Format: Kaci Kopec, Tara Jarboe, Nicole R. DeSouza, Humayun K. Islam, Jan Geliebter, Raj K. Tiwari. Differentiation driver gene HOXD4 as a potential prognostic indicator and therapeutic target in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4633.

Abstract 5668: Long non-coding RNA LINC01614 knockdown decreases carcinogenic behavior of papillary thyroid cancer in vitro

Abstract Papillary thyroid cancer (PTC) accounts for the vast majority of thyroid cancer cases with increasing incidence over the past few decades. PTC has a significantly higher prevalence in females in their childbearing years. Despite high survival rates, disease recurrence and metastasis remain prominent issues, thus highlighting the critical need for molecular biomarker detection. A newer class of molecular regulators, known as long non-coding RNAs (lncRNAs), have demonstrated differential expression patterns in a multitude of cancers. The largest subclass of these RNA molecules in the human genome consists of long intergenic non-coding RNAs (lincRNAs) with many of their functions yet to be identified. Known functional roles of lncRNAs center on their ability to modulate gene expression and drive carcinogenic behavior, deeming them attractive biomarkers and/or therapeutic targets for study. Bioinformatic analysis of RNA sequencing data from our patient sample biobank identified LINC01614 as being significantly upregulated (~12 fold increase) in PTC, when compared to normal, matched thyroid tissue. Further, it was also upregulated ~30-fold in male PTC compared to ~5.5 fold increase in female PTC, highlighting a potential sex correlation for study. Thus, we are studying LINC01614 as a potential PTC biomarker. Gene expression analysis of LINC01614 in vitro was found to be upregulated in various thyroid cancer cell lines, specifically two PTC cell lines: TPC1 (~5 fold; RET/PTC rearrangement; female) and K1 (~2 fold; BRAFV600E; male) when compared to the immortalized “normal” thyroid cell line (Nthy-ori-3-1). For phenotypic evaluation, we utilized the CRISPRi technique to employ transcriptional repression of LINC01614 in both TPC1 and K1. LINC01614 knockdown in TPC1 and K1 resulted in decreased healing capacity (~20%), clonogenicity (~50% and ~34%, respectively), and proliferation (~55% and ~45%, respectively). Studies are currently underway to further assess invasion, morphology, and RNA localization within the cell in vitro, as well as the identification of potential protein interactions and mechanisms of action of LINC01614 in PTC. Studying lncRNAs and their regulatory roles in cancer cell function is critical in the identification of various diagnostic and prognostic biomarkers for therapeutic intervention. Our data support the potential biological role of LINC01614 in driving PTC growth and metastatic potential. Further elucidation of how LINC01614 impacts PTC development and progression is a promising avenue of exploration. Citation Format: Danielle Quaranto, Michelle Carnazza, Nicole R. DeSouza, Sina Dadafarin, Augustine Moscatello, Humayun K. Islam, Raj K. Tiwari, Jan Geliebter. Long non-coding RNA LINC01614 knockdown decreases carcinogenic behavior of papillary thyroid cancer in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5668.

Abstract 7059: Remodeling of anaplastic thyroid cancer metabolic signature by natural alkaloid berberine

Abstract Anaplastic thyroid cancer (ATC) is a rare, fatal cancer with a five-year survival of 4%. Universally diagnosed at stage IV, ATC is characterized by its lack of differentiation and metabolic dysregulation. Refractory to all established therapies, we propose natural alkaloid berberine (BBR) as a therapeutic with multitarget efficacy to alter mitochondrial metabolism and reprogram ATC’s aggressive phenotype. Assays used monocyte cell line U937, ATC cell lines T238 and SW1736, and immortalized normal thyroid cell line Nthy-ori-3-1. All assays used 100µM concentration of BBR or DMSO vehicle control for 24 hours for a tumoricidal effect. Validation of in vitro findings via RNA-Seq was conducted by Genewiz from Azenta and Qiagen’s Ingenuity Pathway Analysis was used for in silico modeling. Mitochondria were isolated via differential centrifugation. Modeling the ATC tumor microenvironment, U937 cells were activated and polarized into a proinflammatory macrophage phenotype. BBR treatment at the activation/polarization stages, 33 soluble inflammatory mediators were downregulated in the conditioned media compared to controls. In targeting the aggressiveness of anaplastic thyroid disease, BBR slowed proliferation by 80% selectively in ATC cells from 48 to 72 hours, while sparing normal cells. BBR reduced migratory capacity by 30% in ATC cells after 24, 48, and 72 hours and reduced migration and invasion by 30%. These observations were substantiated by Western blot analysis - BBR selectively decreased phosphorylation of MEK, ERK, and ribosomal protein S6, crucial downstream regulators of the pro-proliferative and pro-survival pathways in ATC cells. Further, BBR specifically modulated cancer-associated metabolism as observed through an increase in AMPKα phosphorylation, a major rate-limiting protein in cancer-induced dysregulation with an anti-tumor effect. Validation of in vitro findings via RNA-Seq revealed more than 400 significantly differentially expressed genes involved in mitochondrial metabolism, glycometabolism, sirtuin signaling, apoptosis, and proliferation. Following a comprehensive analysis, we identified significant downregulation of 25 of 37 total mitochondrially encoded genes (MTs) and 13 of 13 mitochondrially encoded protein-coding genes comprising the oxidative phosphorylation complexes illuminating a clear link between BBR treatment and altered mitochondrial metabolism in ATC. Additionally, protein expression of significantly downregulated mitochondrial genes identified via RNA Seq was validated via Western blot demonstrating decreased mitochondrially-encoded protein expression related to oxidative phosphorylation. This work reveals a novel role for BBR as an inhibitor of mitochondrial metabolism that can be used to reprogram the aggressive nature of ATC and open the door for promising combination therapy in the treatment of ATC. Citation Format: Tara Jarboe, Nicole R. DeSouza, Kaci Kopec, Jan Geliebter, Xiu-Min Li, Raj K. Tiwari. Remodeling of anaplastic thyroid cancer metabolic signature by natural alkaloid berberine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7059.

Genetic and Environmental Factors in Autoimmune Thyroid Disease: Exploring Associations with Selenium Levels and Novel Loci in a Latvian Cohort.

The interplay of genetic, immune and environmental factors strongly contributes to the development of autoimmune thyroid disease (AITD), which can be classified as Graves' disease (GD) or Hashimoto thyroiditis (HT). One of the most studied exogenous factors in the pathogenesis of AITD is selenium, which, in the form of selenoproteins, strengthens the antioxidative defence system of thyroid cells against superoxide production. Furthermore, it modulates inflammatory cytokine release and autoantibody production. The aim of this study was to assess the associations of genetic factors with selenium levels in a cohort of adults with HT and GD and healthy controls from Latvia. A total of 148 GD patients, 102 HT patients and 2442 control participants were included in the study. The genotypes were determined using genome-wide genotyping; imputation was carried out using the TOPMed r2 imputation panel; and association analysis was performed with PLINK v1.90b7. We found three loci associated with GD (LSAMP, HNRNPA3P5, and NTN1) and one locus associated with HT (VAT1L); furthermore, one locus was associated with a serum selenium concentration > 80 µg/L (LINC01544/RNF152/PIGN). The detected associations could be attributed to population-specific effects or unknown stratification in our cohort, and further assessment of these results is required to explain the relationships of genetic traits with AITD and other phenotypes.

GSG2 promotes thyroid cancer via stabilizing AURKB and activating AKT pathway.

Thyroid cancer stands out as the most prevalent endocrine cancer, with its incidence on a global rise. While numerous studies have delved into the roles of GSG2 in the progression of various malignancies, its involvement in thyroid cancer remains relatively unexplored. Therefore, this study was initiated to assess the functional importance of GSG2 in human thyroid cancer development. Our findings revealed a notable upregulation of GSG2 in both thyroid cancer tissues and cell lines, demonstrating a significant correlation with the pathological stage and patients' prognosis. Depletion of GSG2 in thyroid cancer cells resulted in suppressed malignant cell development and inhibited tumor outgrowth. Crucially, our investigation identified AURKB as a downstream gene of GSG2. GSG2 exhibited its regulatory role by stabilizing AURKB, countering SMURF1-mediated ubiquitination of AURKB. Furthermore, overexpressing AURKB restored the functional consequences of GSG2 depletion in thyroid cancer cells. Additionally, we proposed the involvement of the AKT pathway in GSG2-mediated regulation of thyroid cancer. Intriguingly, the reversal of cell phenotype alterations in GSG2-depleted cells following an AKT activator underscored the potential link between GSG2 and the AKT pathway. At the molecular level, GSG2 knockdown downregulated p-AKT, an effect partially restored after AKT activator treatment. In summary, our study concluded that GSG2 played a pivotal role in thyroid carcinogenesis, underscoring its potential as a therapeutic target for thyroid cancer.