Normal Thyroid Cells Research Articles

A new LNC89/LNC60-Col11a2 axis revealed by whole-transcriptome analysis may be associated with goiters related to excess iodine nutrition

Goiter related to excessive iodine nutrition remains a significant public health issue in some countries. There has been no reported study on long noncoding RNAs (lncRNAs) related to goiters. In this study, goiter was induced by drinking water with excess iodine for 10 or 20 weeks in Kunming mice. Whole transcriptome sequencing results showed that LNC89 expression increased in mice goiter tissues compared to normal thyroid tissues and higher in 20 weeks goiter tissues than in 10 weeks goiter tissues, which were identified by qRT−PCR. Cooperate with human-mouse homologous gene conversion, a new LNC89/LNC60-Col11a2 axis was predicted by LncTar and expression correlation analysis based on whole transcriptome sequencing results. Increased Col11a2 expression was also identified by qRT−PCR and Western blot in the mice goiter tissues. In the human normal thyroid cell line Nthy-ori-3 treated with KI03, LNC60 and Col11a2 expression increased with promoted cell viability, which were reversed by siLNC60 treatment. Furthermore, LNC60 and Col11a2 mRNA levels were found increased in peripheral blood of nodular goiter patients from high water iodine areas of China and have high diagnostic values for nodular goiter while AUC of LNC60 and Col11a2 are 89.97% and 84.85%, respectively. In conclusion, the novel LNC89/LNC60-Col11a2 axis may be involved in the progression of goiter related to iodine excess, providing potential biomarkers and therapeutic targets in the future.

PFAS-mediated disruptions on thyroid histology

Abstract Introduction/Objective Per-and polyfluroalkyl substances (PFAS), are environmental contaminants of emerging concern due to their abundance in the environment and potential adverse health impacts. PFAS are used in waterproof clothing, makeup, carpets, upholstery, cookware, and fast-food containers. These organic pollutants have been found in the blood of 98% of Americans and have been linked to disruption in thyroid hormones. During fetal life, thyroid hormones are crucial for brain development and alterations in these hormones can induce long-term impacts on intellectual and brain impairments. T3 and T4 are known to be altered by PFAS exposure, but the mechanism by which this is done remains unclear. Methods/Case Report Normal thyroid cells were treated for 72 hours with 1000 nM solution containing either GEnX, PFOA, PFOS, or 1% DMSO control. Immunofluorescence for actin, tubulin, and DAPI was performed using a Nikon spinning disk. In vivo studies where 8-week-old mice are gavaged with 7.5 mg/kg of PFOS, PFOA, and GenX or PBS control for 8 weeks. Results (if a Case Study enter NA) We hypothesized the PFAS treatment alters thyroid cells morphology and histology. Immunofluorescence staining of normal thyroid cells treated with PFAS show a decrease in actin stress fibers compared to control cells. PFAS treated mice show a decrease in follicle size compared to control (average 1017 um2 size, p=0.0483). Conclusion This is the first study to show changes in thyroid histology after 8 weeks of PFAS exposure. As structure and function are closely linked, it is likely that this reduction in follicle size and actin reorganization contribute to alterations in thyroid function. Future studies are needed to illuminate the mechanism by which PFAS alter thyroid function in vivo. Understanding the role of PFAS-mediated thyroid dysregulation will have far reaching implications on environmental regulations and considerations for limiting PFAS in our environment.

Quercetin and Thyroid.

Quercetin is the most abundant flavonoid present in fruits and vegetables. For its antiproliferative, antiviral, anti-inflammatory and antioxidants activities, it is an active ingredient of several herbal remedies and is available as a nutraceutical. Experimental studies performed in vitro have demonstrated that quercetin inhibits growth and function in normal thyroid cells and may act as a thyroid disruptor. These effects have also been confirmed in vivo using rodent models. Some studies have reported the ability of quercetin to interfere with the metabolism of thyroid hormones, since it inhibits the 5'-deiodinase type 1 (D1) activity in the thyroid, as well as in the liver. Besides the effects on normal thyroid cells, several experiments performed in vitro have shown a potential therapeutic role of quercetin in thyroid cancer. Indeed, quercetin inhibits the growth, the adhesion and the migration of thyroid cancer cells, and it also has redifferentiation properties in some thyroid cancer cell lines. In conclusion, these data suggest that, although its effects can be of benefit in hyperthyroidism and thyroid cancer, caution is required in the use of high doses of quercetin due to its anti-thyroid properties. Further in vivo studies are certainly needed to confirm these hypotheses.

Neutrophil elastase specific fluorescent probe for early diagnosis of thyroiditis via serum sample testing and fluorescence imaging

Autoimmune thyroid diseases (AITD) often interfere with early detection due to asymptomatic symptoms and normal thyroid function in routine tests. Developing early diagnostic tools is crucial for timely and accurate treatment, potentially reducing complications and improving patient outcomes. In this study, we developed Ox-NE, a novel fluorescent probe designed for the specific detection and quantification of neutrophil elastase (NE), a key biomarker of inflammation. Unlike the traditional probes, Ox-NE utilizes a unique mechanism that minimizes background fluorescence and enhances photostability, offering rapid, non-invasive, and apparent diagnostic capabilities. Ox-NE had a low limit of detection (LOD) of 1.54 μg/mL and exhibited high sensitivity and specificity with strong anti-interference properties. Through in vitro experiments, Ox-NE could accurately detect elevated NE levels in the serum of thyroiditis patients. Additionally, it could differentiate between normal thyroid cells, inflamed thyroid cells, and thyroid cancer cells. It also effectively facilitated the screening of sivelestat, a therapeutic agent for thyroiditis. Successful fluorescence imaging in mouse models further confirmed the potential of Ox-NE in advancing thyroid disease diagnostics.

Selective anti-tumor activity of glutathione-responsive abasic site trapping agent in anaplastic thyroid carcinoma

Anaplastic thyroid carcinoma (ATC) is a rare but highly aggressive thyroid cancer with poor prognosis. Killing cancer cells by inducing DNA damage or blockage of DNA repair is a promising strategy for chemotherapy. It is reported that aldehyde-reactive alkoxyamines can capture the AP sites, one of the most common DNA lesions, and inhibit apurinic/apyrimidinic endonuclease 1(APE1)-mediated base excision repair (BER), leading to cell death. Whether this strategy can be employed for ATC treatment is rarely investigated. The aim of this study is to exploit GSH-responsive AP site capture reagent (AP probe-net), which responses to the elevated glutathione (GSH) levels in the tumor micro-environment (TME), releasing reactive alkoxyamine to trap AP sites and block the APE1-mediated BER for targeted anti-tumor activity against ATC. In vitro experiments, including MTT andγ-H2AX assays, demonstrate their selective cytotoxicity towards ATC cells over normal thyroid cells. Flow cytometry analysis suggests that AP probe-net arrests the cell cycle in the G2/M phase and induces apoptosis. Western blotting (WB) results show that the expression of apoptotic protein increased with the increased concentration of AP probe-net. Further in vivo experiments reveal that the AP probe-net has a good therapeutic effect on subcutaneous tumors of the ATC cells. In conclusion, taking advantage of the elevated GSH in TME, our study affords a new strategy for targeted chemotherapy of ATC with high selectivity and reduced adverse effects.

Iron oxide nanoparticles of Cystoseira sp. Sugar alcohol treat MRSA and thyroid gland cancer

PurposeMethicillin-resistant Staphylococcus aureus (MRSA) infections are emerging as a cause of suppurative thyroiditis. The resistance to several antibiotics and finding treatment has grown more difficult. Hence, the purpose of this study was to examine biosynthesized iron oxide nanostructures’ antimicrobial activity as well as their cytotoxicity on normal and thyroid cancer cells. MethodsBy using the sugar alcohols of Cystoseira sp., the authors synthesized the advanced biosynthesis of iron oxide nanoparticles (COINs). An agar-well diffusion method and a broth dilution method were used to investigate COINs’ antimicrobial activity against MRSA isolates. However, their cytotoxicity against normal and thyroid cancer cell lines was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. ResultsA GC–MS analysis of the dried Cystoseira sp. extract revealed a high percentage of D-mannitol, which is essential for the synthesis of spherical nanoparticles with a mean average size of 5.8 ± 0.8 nm. The COINs showed a higher inhibition zone (9–15 mm) for 66.6 % of MRSA isolates, and the MIC of COINs was 256 μg/ml. The cell wall of these bacteria inhabits the absorption of COINs. As a result, a large concentration of COINs is required to restrict bacterial growth. Moreover, COINs have cytotoxicity on thyroid cancer cell proliferation and normal thyroid cell lines at IC50s of 1.71 ± 0.1 and 25.9 ± 1.6 μg/ml, respectively. This can be due to the negative charge of sugar alcohols, which affect protein absorption and subsequent biological behaviors. ConclusionThe promising findings of this study are in favor of the creation of very small iron oxide nanostructures for therapy.

KLHL14 is a tumor suppressor downregulated in undifferentiated thyroid cancer

KLHL14 is a substrate-binding subunit of Cullin-RING ligase 3 ubiquitin ligase complex, highly enriched in thyroid since early embryonic development, together with its antisense RNA KLHL14-AS. We have previously demonstrated that Klhl14-AS is a competing endogenous RNA regulating several differentiation and survival factors in thyroid cancer, acting as tumor suppressor. Recently, also KLHL14 has been shown to function as tumor suppressor in diffuse large B-cell lymphoma and in malignant mesothelioma. Here we show that KLHL14 expression is strongly reduced in anaplastic thyroid cancer, the less differentiated and most aggressive type of thyroid neoplasia. Such reduction is reproduced in different in vivo and in vitro models of thyroid cancer, being invariably associated with loss of differentiation. When Klhl14 expression is rescued in thyroid transformed cells, it reduces the cell proliferation rate and increase the number of apoptotic cells. On the other side, Klhl14 loss of function in normal thyroid cells affects the expression of several regulatory as well as functional thyroid markers. All these findings suggest that KLHL14 could be considered as a novel tumor suppressor in thyroid cancer, by also revealing its physiological role in the maintenance of a fully differentiated and functional thyroid phenotype.

The common pathogenesis of nodular goiter in both sexes: An exploration into gene expression and signaling pathways

The past few years have witnessed an increasing incidence of nodular goiter (NG), with a well-documented higher prevalence in females than males. This gender disparity has led research to focus primarily on female subjects, potentially overlooking common pathogenic mechanisms in both sexes. In this study, we investigated the shared pathogenesis of NG in males and females. Utilizing a rat model and RNA sequencing, we identified differentially expressed genes associated with the disease. We further validated these findings in normal human thyroid cells and human papillary thyroid cancer cells. A randomized experiment was conducted with equal numbers of male and female rats divided into control and NG model groups. The NG model was established using propylthiouracil and various assessments such as thyroid ultrasonography, thyroid index, thyroid function, and thyroid histology were performed. Transcriptome analysis revealed numerous upregulated and downregulated genes in both male and female model groups. Key genes like KDR, FLT1, PDGFB, and CAV1, and pathways including PI3K-Akt, MAPK, Ras, fluid shear stress and atherosclerosis, calcium signaling, and Rap1 signaling pathways were linked with the disease. Western blot and immunofluorescence analysis confirmed these findings, which were further supported by cell-based experiments. In conclusion, our findings suggest that abnormal expression of specific genes and pathways leading to irregular cell growth, blood vessel formation, and inflammation may be common factors in the pathogenesis of NG in both males and females.

Selective anti-CXCR2 receptor blockade by AZD5069 inhibits CXCL8-mediated pro-tumorigenic activity in human thyroid cancer cells in vitro.

Thyroid cancer is the most common endocrine malignancy. Current therapies are successful, however some patients progress to therapeutically refractive disease. The immunotherapeutic potential of the CXCL8-chemokine/CXCR2-chemokine-receptor system is currently being explored in numerous human cancers. This study aimed to evaluate if the targeting of CXCR2 by its selective antagonist, AZD5069, could modulate CXCL8-mediated pro-tumorigenic effects in thyroid-cancer (TC) cells in vitro. Normal human primary thyroid cells (NHT) and TC cell lines TPC-1 (RET/PTC), BCPAP, 8505C and 8305C (BRAFV600e) were treated with AZD5069 (100pM-10µM) over a time-course. Viability and proliferation were assessed by WST-1 and crystal violet assays. CXCL8 and CXCR2 mRNA were evaluated by RT-PCR. CXCL8-protein concentrations were measured in cell culture supernatants by ELISA. CXCR2 on cell surface was evaluated by flow-cytometry. Cell-migration was assessed by trans-well-migration chamber-system. AZD5069 exerted negligible effects on cell proliferation or viability. AZD5069 significantly reduced CXCR2, (but not CXCL8) mRNAs in all cell types. CXCR2 was reduced on the membrane of some TC cell lines. A significant reduction of the CXCL8 secretion was found in TPC-1 cells (basal-secretion) and NHT (TNFα-induced secretion). AZD5069 significantly reduced basal and CXCL8-induced migration in NHT and different TC cells. Our findings confirm the involvement of the CXCL8/CXCR2-axis in promoting pro-tumorigenic effects in TC cells, further demonstrating its immunotherapeutic significance in human cancer.

A Specificity Protein 1 assists the Progression of the Papillary Thyroid Cell Line by Initiating NECTIN4.

Papillary thyroid cancer (PTC) is one of the subtypes of thyroid cancer with increasing incidence worldwide, but the molecular mechanism is still unclear. Papillary thyroid cancer (PTC) is one of the subtypes of thyroid cancer with increasing incidence worldwide, but the molecular mechanism is still unclear. Studies have indicated that nectin cell adhesion molecule 4 (NECTIN4) was an oncogene and played an important role in the development and progression of PTC. Meanwhile, specificity protein 1 (SP1) expresses many important oncogenes and tumor suppressor genes. However, the relationship between NECTIN4 and SP1 in regulating PTC growth is unclear. In the present study, reverse transcription PCR was utilized to detect the mRNA expression of NECTIN4 and SP1 in thyroid cancer cell lines and normal thyroid cell lines. Chromatin immunoprecipitation assays and luciferase reporter assays were used to study whether SP1 could bind to the promoter region of NECTIN4 and activate its transcription. The biological functions of SP1 correlated with NECTIN4 were also performed in TPC-1 and KTC1 cell lines. The study revealed that the mRNA expression level of SP1 and NECTIN-4 showed a positive correlation and were upregulated in PTC cell lines. Moreover, the results of ChIP and luciferase reporter assays showed that SP1 could bind to the NECTIN4 promoter regions and activate the transcriptional level of NECTIN4. The experiments in vitro showed that SP1 could promote cell proliferation, colony formation, migration, and invasion by regulating NECTIN4 in PTC cells. In conclusion, our study, for the first time, demonstrated that SP1 could control the transcriptional regulation of NECTIN4 and accelerate the growth of PTC, which may provide a new potential therapeutic target for PTC patients.

Bioinformatic analysis of CCND2 expression in papillary thyroid carcinoma and its impact on immune infiltration

To investigate cyclin D2 (CCND2) expression in papillary thyroid carcinoma (PTC) and its association with the clinicopathological features. The public databases TCGA, TIMER 2.0 and UALCAN were used to explore CCND2 expression level in PTC and adjacent tissues, and its diagnostic value for PTC was analyzed using ROC curves. GO enrichment analysis of CCND2-related differentially expressed genes (DEGs) in PTC was performed, and tumor immune infiltration of CCND2 in thyroid cancer was analyzed using TIMER database and CIBERSORT data source. RT-qPCR and Western blot were used to detect CCND2 expression in normal human thyroid cell line Nthy-ori-3-1 and human PTC cell lines TPC-1 and BCPAP. CCND2 expression was also detected in clinical specimens of PTC and adjacent tissues by immunohistochemistry, and its correlation with clinicopathological features of the patients were analyzed. Informatic analysis revealed significantly higher CCND2 mRNA expression in thyroid cancer than in the adjacent tissues (P < 0.001) in close correlation with tumor stage, gender, age, pathological subtype, and lymph node involvement (P < 0.05). ROC curve analysis showed that at the cutoff value of 4.983, the diagnostic sensitivity, specificity, and accuracy of CCND2 expression for PTC was 83.6%, 94.9%, and 78.5%, respectively. CCND2 expression was positively correlated with B cells, CD4+ T cells, and macrophages (P < 0.001) and negatively with CD8+ T cells (P < 0.01), and also correlated with memory B-cell infiltration, CD4+ T-cell memory activation, M2 macrophages, resting mast cells, and mast cell activation (P < 0.05). RT-qPCR, Western blot and immunohistochemistry showed significantly higher CCND2 expression in the PTC cells than in Nthy-ori-3-1 cells (P < 0.01) and also in clinical PTC tissues than in the adjacent tissues (P < 0.05) in correlation with tumor size, lymph node metastasis and TNM stage (P < 0.05). CCND2 overexpression is closely correlated with tumor progression and immune cell infiltration in PTC patients..

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.

Investigation of dextran-coated magnetic nanoparticles encapsulated by medication and modified with folate for targeted drug delivery: Invitro 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.

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.

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.

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 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.

LncRNA HCG22 regulated cell growth and metastasis of papillary thyroid cancer via negatively modulating miR-425-5p.

Papillary thyroid cancer (PTC) is a common malignant tumour in the endocrine system with increasing incidence. LncRNA HCG22 (HCG22) was noticed to be dysregulated in PTC, but its specific function and mechanism remain unknown. The function of HCG22 and its underlying molecular mechanism was investigated to evaluate its potential as a biomarker for PTC. The expression of HCG22 was detected in PTC cells (TPC-1, SNU790, GLAG-66, and BCPAP) and normal thyroid cells (Nthy-ori) using real time quantative polymerase chain reaction (RT-qPCR). HCG22 and miR-425-5p were regulated by cell transfection. The cell proliferation and metastasis were assessed by CCK8 and Transwell assay. HCG22 was upregulated in PTC cells, of which the knockdown suppressed the proliferation, migration, and invasion of PTC cells. miR-425-5p was downregulated in PTC cells, which was negatively regulated by HCG22. Silencing miR-425-5p could reverse the inhibitory effect of HCG22 knockdown on the cellular processes of PTC. HCG22 served as a tumour promoter in PTC cells, which regulated cell proliferation and metastasis via negatively regulating miR-425-5p.

Identification of Germline FOXE1 and Somatic MAPK Pathway Gene Alterations in Patients with Malignant Struma Ovarii, Cleft Palate and Thyroid Cancer.

Germline variants in the FOXE1 transcription factor have been associated with thyroid ectopy, cleft palate (CP) and thyroid cancer (TC). Here, we aimed to clarify the role of FOXE1 in Portuguese families (F1 and F2) with members diagnosed with malignant struma ovarii (MSO), an ovarian teratoma with ectopic malignant thyroid tissue, papillary TC (PTC) and CP. Two rare germline heterozygous variants in the FOXE1 promoter were identified: F1) c.-522G>C, in the proband (MSO) and her mother (asymptomatic); F2) c.9C>T, in the proband (PTC), her sister and her mother (CP). Functional studies using rat normal thyroid (PCCL3) and human PTC (TPC-1) cells revealed that c.9C>T decreased FOXE1 promoter transcriptional activity in both cell models, while c.-522G>C led to opposing activities in the two models, when compared to the wild type. Immunohistochemistry and RT-qPCR analyses of patients' thyroid tumours revealed lower FOXE1 expression compared to adjacent normal and hyperplastic thyroid tissues. The patient with MSO also harboured a novel germline AXIN1 variant, presenting a loss of heterozygosity in its benign and malignant teratoma tissues and observable β-catenin cytoplasmic accumulation. The sequencing of the F1 (MSO) and F2 (PTC) probands' tumours unveiled somatic BRAF and HRAS variants, respectively. Germline FOXE1 and AXIN1 variants might have a role in thyroid ectopy and cleft palate, which, together with MAPK pathway activation, may contribute to tumours' malignant transformation.

First Evidence of Mineralocorticoid Receptor Gene and Protein Expression in Rat and Human Thyroid Tissues and Cell Cultures.

Aldosterone (Aldo) exerts its action through binding with the mineralocorticoid receptor (MR). Clinically, a link between primary aldosteronism (PA) and thyroid diseases has been hypothesised. However, the presence and activity of MR on the thyroid have not yet been demonstrated. We investigated the gene/protein expression and activation of MR in primary thyroid cell cultures (normal rat thyroid [FRTL-5] and human papillary thyroid cancer [PTC] cell lines, BCPAP and K1) through qRT-PCR analysis, immunofluorescence, and confocal microscopy. We also studied the effects of Aldo on thyroid-specific and inflammation genes in vitro. Paired human normal and neoplastic thyroid tissues were also studied. We demonstrated both gene and protein expression and activation of MR in normal rat thyroid and human PTC lines. Incubation with Aldo induced an acute increase in IL-6 expression in both the FRTL-5 and BCPAP lines, which was antagonised by spironolactone, and an acute and late upregulation of thyroid-specific genes in FRTL-5. MR was also expressed at both gene and protein levels in normal human thyroid tissues and in PTC, with a progressive decline during neoplastic tumourigenesis, particularly in more aggressive histotypes. We present the first evidence of MR gene and protein expression in both normal and pathological thyroid cells and tissues. We have shown that MR is present and functionally activated in thyroid tissue. Binding of Aldo to MR induces the expression of inflammatory and thyroid-specific genes, and the thyroid may thus be considered a novel mineralocorticoid target tissue.