TPC-1 Cells Research Articles

PFOA, PFHxA and C6O4 differently modulate the expression of CXCL8 in normal thyroid cells and in thyroid cancer cell lines.

Industrial chemical PFAS are persistent pollutants. Long chain PFAS were taken out of production due to their risk for human health, however, new congeners PFAS have been introduced. The in vitro effects of the long-chain PFOA, the short-chain PFHxA and the new-generation C6O4 were evaluated in normal and in thyroid cancer cell lines in terms of cell viability and proliferation, and secretion of a pro-tumorigenic chemokine (CXCL8), both at the mRNA and at the protein level. The Nthy-ory 3-1 normal-thyroid cell line, the TPC-1 and the 8505C (RET/PTC rearranged and BRAFV600e mutated, respectively) thyroid-cancer cell lines were exposed to increasing concentrations of each PFAS in a time-course. We evaluated viability using WST-1 (confirmed by AnnexinV/PI) and proliferation using the cristal-violet test. To evaluate CXCL8 mRNA we used RT-PCR and measured CXCL8 in the supernatants by ELISA. The exposure to none PFAS did not affect thyroid cells viability (except for a reduction of 8505C cells viability after 144h) or proliferation. Individual PFAS differently modulated CXCL8 mRNA and protein level. PFOA increased CXCL8 both at mRNA and protein level in the three cell lines; PFHxA increased CXCL8 mRNA in the three cell lines, but increased the protein only in TPC-1 cells; C6O4 increased the CXCL8 mRNA only in thyroid cancer cell lines, but never increased the CXCL8 protein. The results of the present study indicate that the in vitro exposure to different PFAS may modulate both at the mRNA and secreted protein levels of CXCL8 in normal and cancer thyroid cells. Strikingly different effects emerged according to the specific cell type and to the targeted analyte (CXCL8 mRNA or protein).

TESC promotes differentiated thyroid cancer development by activating ERK and weakening NIS and radioiodine uptake.

Most differentiated thyroid cancer (DTC) patients have a good prognosis after surgery, but radioiodine refractory differentiated thyroid cancer (RAIR-DTC) patients have a significantly reduced 5-year survival rate (<60%) and a significantly increased recurrence rate (>30%). This study aimed to clarify the tescalcin (TESC) role in promoting the malignant PTC progression and providing a potential target for RAIR-DTC treatment. We analyzed TESC expression and clinicopathological characteristics using the Cancer Genome Atlas (TCGA) and performed qRT-PCR on tissue samples. TPC-1 and IHH-4 proliferation, migration, and invasion were detected after transfection with TESC-RNAi. Using Western blot (WB), several EMT-related indicators were detected. Moreover, iodine uptake of TPC-1 and IHH-4 after transfection with TESC-RNAi was detected. Finally, NIS, ERK1/2, and p-ERK1/2 levels were determined by WB. TESC was significantly upregulated in DTC tissues and positively correlated with BRAF V600E mutation based on data analysis from TCGA and our center. Reduced expression of TESC in both IHH-4 (BRAF V600E mutation) and TPC-1 (BRAF V600E wild type) cells significantly inhibited cell proliferation, migration, and invasion. It downregulated the EMT pathway markers Vimentin and N-cadherin, and increased E- cadherin. Moreover, TESC knockdown significantly inhibited ERK1/2 phosphorylation and decreased NIS expression in DTC cells, with a remarkably increased iodine uptake rate. TESC was highly expressed in DTC tissues and may have promoted metastasis through EMT and induced iodine resistance by downregulating NIS in DTC cells.

Abstract 1405: Long non-coding RNA LINC01614 augments papillary thyroid cancer cell phenotype

Abstract Papillary thyroid cancer (PTC) accounts for the vast majority of thyroid cancer cases and despite high survival rates, disease recurrence and metastasis remain prominent issues. Therefore, there is a need to identify potential molecular markers associated with PTC that aid in early diagnosis, prognosis, and that may serve as therapeutic targets. One area of focus for cancer study is trending towards the epigenome and its functional regulators. The largest subclass of RNA molecules in the human genome consists of long intergenic non-coding RNAs (lincRNAs) with many of their functions yet to be identified. Evidence supports their roles in the regulation of gene expression, as these molecules can act as scaffolds for RNA and protein localization as well as act as molecular “sponges” for binding to and inhibiting action of specific miRNAs. Recently, long non-coding RNA molecules have been demonstrated to play roles in both the development and progression of cancer, deeming them as both attractive and specific biomarkers and/or therapeutic targets for study. Data from our patient sample biobank identified LINC01614 as being significantly upregulated (~12 fold increase) in PTC, when compared to normal, matched thyroid tissue. There was also ~30 fold increase in LINC01614 expression 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. Functional analysis of LINC01614 in vitro found it 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). To establish a phenotype for LINC01614 expression, we utilized the CRISPRi technique to knockdown expression of this lincRNA in both TPC1 and K1. Knockdown resulted in decreased healing capacity (~20% for both), clonogenicity (~50% and ~34%, respectively), and proliferation (~55% and ~45%, respectively) in both the TPC1 and K1 PTC cell lines. Studies are underway to further assess migration, invasion, and morphology in both cell lines as well as studies to elucidate potential protein interactions and mechanisms of action of LINC01614 in PTC. Studying lincRNAs and their regulatory roles in cancer cell function can aid in the identification of various molecular markers for therapeutic intervention. Elucidation of how these molecules impact PTC development and progression is a promising avenue of exploration. Citation Format: Danielle Quaranto, Michelle Carnazza, Nicole DeSouza, Sina Dadafarin, Augustine Moscatello, Raj Tiwari, Jan Geliebter. Long non-coding RNA LINC01614 augments papillary thyroid cancer cell phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1405.

Abstract 3946: Radiolabeled αvβ3 analog 177Lu-EB-RGD is an effective therapeutic agent in thyroid cancer xenograft mouse model

Abstract Background: Integrins are cell adhesion receptors consisting of 18α and 8β subunits. A subset of integrins (αvβ3) recognizes Arg-Gly-Asp (RGD) peptide motifs which are involved in the neovascularization and progression of various cancers. The aim of the study was to investigate if αvβ3 can serve as a molecular target for the treatment of thyroid cancer (TC) with a novel radiolabeled RGD analog 177Lu-EB-RGD. Methods: Integrin αvβ3 mRNA and/or protein expression was evaluated in 496 TC included in The Cancer Genome Atlas, tissue microarray including 70 TC and 10 normal thyroid samples, and 14 TC cell lines. BRAF-like or RAS-like expression profile was determined through standard BRS scores ranging from -1 to 0 for BRAF-like and 0 to 1 for RAS-like TC. The association between BRS and the αvβ3 expression was tested by the Spearman correlation (r). Nude mice xenografts developing αvβ3 expressing TC after subcutaneous injection of 5*10^6 TC cells were subjected to monotherapy with 0.5 mCi 177Lu-EB-RGD (177Lu) or in combination with Lenvatinib (177Lu+L). The therapeutic efficacy of 177Lu-EB-RGD was compared with standard-of-care Lenvatinib alone (L) and placebo (P). The continuous data were presented as medians with [25-75% interquartile ranges] and compared using Kruskal-Wallis test. The mixed-effects models were used for longitudinal data analysis with adjusted p≤ 0.05 considered statistically significant. Results: We found a moderate negative correlation between BRS and αv (r=-0.5, p&amp;lt;0.001), and β3 (r=-0.27, p&amp;lt;0.001), revealing that BRAF-like tumors have a higher mRNA expression of αvβ3 integrins. Consistently, the highest αvβ3 mRNA and/or protein expression was found in the BRAF-like TC cell lines OCUT2 (BRS=-0.56), TPC1 (BRS=-0.4), K1 (BRS=-0.29), and Hurthle TC cell line XTC1 (BRS=-0.46). The immunostaining revealed a higher αvβ3 expression in papillary TC compared with follicular TC (p=0.002), and in normal thyroid (p&amp;lt;0.001). Poorly differentiated TC had a similar αvβ3 expression to papillary TC (p=0.14). In the thyroid cancer xenograft mouse model, all treatment modalities were more effective than the placebo in decreasing tumor size as early as 5 days after therapy initiation. A significant difference in growth curve and tumor volume was maintained at the study endpoint (L 0.584 cm3 [0.196-0.984] vs P 0.911 cm3 [0.183-1.68], p=0.001; 177Lu 0.259 cm3 [0.103-0.376] vs P, p&amp;lt;0.001; and 177Lu+L 0.274 cm3 [0.108-0.406] vs P, p&amp;lt;0.001). The combination therapy (177Lu+L) resulted in decreased tumor volume as compared with monotherapy with Lenvatinib (p=0.05) but had a similar effect as compared with 177Lu-EB-RGD monotherapy (p=0.99). Conclusions: The radiolabeled αvβ3 analog 177Lu-EB-RGD has potent growth inhibitory effects in TC characterized by a high integrins expression. The αvβ3 integrin could potentially serve as a molecular target for therapy with radiolabeled RGD analogs in TC. Citation Format: Sonam Kumari, Zhantong Wang, Shilpa Thakur, Laura Abaandou, Oksana Gavrilova, Huiyan Lu, Lixin Lang, Dale Kiesewetter, Vasyl Vasko, Joanna Klubo-Gwiezdzinska. Radiolabeled αvβ3 analog 177Lu-EB-RGD is an effective therapeutic agent in thyroid cancer xenograft mouse model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3946.

EF-24, a Curcumin Analogue, Could Inhibit HSP90 and Induce Apoptosis-Mediated by Autophagy/Inflammation/Oxidative Stress in Papillary Thyroid Cancer Cell Lines

Objective Papillary thyroid cancer (PTC) is one of the most common thyroid cancers and its prevalence has increased in recent years. Studies have shown that autophagy, angiogenesis, inflammation, oxidative damage, apoptosis, and some signaling pathways such as PI3K/AKT/mTOR play an important role in cancer progression. Moreover, most of their signaling molecules are the clients of heat shock protein 90 (HSP90), and its inhibition attenuates tumorigenesis. Diphenyl difluoroketone (EF-24), a curcumin analog, has antioxidative, anti-inflammatory, and anticancer properties and, unlike curcumin, has high efficacy, and may treat PTC. Therefore, the antioxidant, anti-inflammatory, angiogenic, apoptotic, and anticancer effects of EF-24 on PTC cell lines (BCPAP and TPC-1) were investigated in this study. Methods In this study, the effect of different concentrations of EF-24 and cisplatin on cell viability, apoptosis, inflammation, autophagy, oxidative status, and angiogenesis, along with HSP90 expression was evaluated in BCPAP and TPC-1 cell lines. Results The results showed that treatment with different EF-24 concentrations could significantly decrease cell viability by inducing apoptosis. EF-24 (40 µg/mL) also significantly downregulated HSP90 and the gene and protein involved in oxidative damage, angiogenesis, inflammation, and autophagy. Conclusions In conclusion, EF-24 could decrease cell viability by inducing apoptosis mediated by autophagy, oxidative stress, angiogenesis, inflammation, and the PI3K/AKT/mTOR pathway. It is possible that a decrease in HSP90 may mediate these effects. These results indicate that EF-24 is a viable option for the prevention and treatment of PTC.

Long non‑coding RNA DSCAM‑AS1 functions as an oncogene in thyroid cancer via regulating miR‑211.

Long non-coding RNA Down syndrome cell adhesion molecule-antisense 1 (DSCAM-AS1) has been reported to play key roles in the progression and initiation of several cancer types. However, the various functional roles of DSCAM-AS1 in thyroid cancer tumorigenesis remain largely elusive. In the present study, the expression of DSCAM-AS1 was examined in thyroid cancer tissues and cell lines. Cell Counting Kit-8, wound healing, Transwell and clonogenic assays were conducted to detect cell proliferation, migration, invasion and colony formation, respectively. The association of DSCAM-AS1 with microRNA 211 (miR-211) was determined by luciferase reporter assay. It was found that the expression of DSCAM-AS1 was upregulated in thyroid cancer cells and tissues. Furthermore, enhanced DSCAM-AS1 expression was positively associated with lymph node metastasis and tumor-node-metastasis stage. Functional experiments demonstrated that DSCAM-AS1 knockdown inhibited the migration, proliferation and invasion of TPC-1 cells. Mechanistically, DSCAM-AS1 could bind to miR-211. Prevention of miR-211 by a miR-211 inhibitor reversed the effect of DSCAM-AS1 depletion in thyroid cancer tumorigenesis. Briefly, the current findings suggested that knockdown of DSCAM-AS1 suppressed the tumorigenesis of thyroid cancer via regulating miR-211, suggesting that DSCAM-AS1 may be a favorable therapeutic target for thyroid cancer.

Circ_0092315 Promotes Proliferation and Invasion of Papillary Thyroid Carcinoma Cells via Regulating microRNA-1256/High Mobility Group A2 axis

Objective To investigate the role and mechanism of circ_0092315 in the proliferation and invasion of papillary thyroid carcinoma cells. Methods The expression of circ_0092315 in papillary thyroid carcinoma cells was examined by real-time fluorescence quantitative PCR.The proliferation and invasion of TPC-1 cells was assessed by CCK-8 and Transwell assays.The protein level of high mobility group A2 (HMGA2) was determined by Western blotting.The regulatory relationship of circ_0092315,microRNA-1256 (miR-1256),and HMGA2 was explored by bioinformatics tools,dual-luciferase reporter assay,real-time fluorescence quantitative PCR,and Western blotting. ++++Results circ_0092315 was overexpressed in papillary thyroid carcinoma cells (all P<0.001).circ_0092315 promoted the proliferation and invasion of TPC-1 cells (all P<0.001).The transfection of si-circ_0092315 up-regulated the expression of miR-1256 (P<0.001),and miR-1256 inhibitor up-regulated the protein level of HMGA2 (P<0.001). ++++Conclusion circ_0092315 is overexpressed in TPC-1 cells and it promotes the proliferation and invasion of TPC-1 cells by regulating the miR-1256/HMGA2 axis.

Effects of miR-145 targeting rab5c and regulating MAPK / ERK signaling pathway on proliferation and invasion of thyroid papillary carcinoma cells.

The objective of this research was to analyze the miR-145 function in thyroid papillary carcinoma cells and explore its possible mechanism. For this purpose, the TPC-1 cell line was selected, miR-145 overexpression and rab5c shRNA lentiviral vector were constructed, and transfected into PTC cells. Luciferase reporter gene was performed to determine the relationship between miR-145 and rab5c, Western blot and qPCR were performed to detach the expression of the related genes, CCK-8 cell proliferation assay and Transwell cell invasion assay were used to determine the proliferation and invasion ability of PTC-1 cells. Results showed that MiR-145 overexpression inhibited the wt-rab5c (wild-type rab5c)luciferase activity, decreased the expression of rab5c mRNA and protein levels in the TPC-1 cell line, inhibited the proliferation and invasion of PTC cell line TPC-1(P < 0.05). In TPC-1 cells, both miR-145 overexpression and RNA interference with rab5c could increase the expression of the p-ERK protein (P < 0.05). In conclusion, MiR-145 inhibits the proliferation and invasion of PTC cells by downregulating rab5c and activating MAPK/ERK pathway in vitro.

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

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

Resolving the role of podoplanin in the motility of papillary thyroid carcinoma-derived cells using RNA sequencing

The intracellular level of podoplanin (PDPN), a transmembrane protein of still unclear function, is frequently altered in metastatic tumors. High expression of PDPN is frequently observed in papillary thyroid cancer (PTC) specimens. Similarly, PTC-derived cell lines (BCPAP and TPC1, harboring the BRAF V600E mutation and RET/PTC1 fusion, respectively), also present enhanced PDPN yield. We previously reported that depletion of PDPN impairs migration of TPC1 cells, but augments metastasis of BCPAP cells. Interestingly, this phenomenon stays in contrast to the migratory pattern observed for wild-type cells, where TPC1 exhibited higher motility than BCPAP cells. Here, we aimed to elucidate the potential role of PDPN in regulation of molecular mechanisms leading to the diverse metastatic features of the studied PTC-derived cells. We consider that this phenomenon may be caused by alternative regulation of signaling pathways due to the presence of the mutated BRAF allele or RET/PTC1 fusion. The high-throughput RNA sequencing (RNA-seq) technique was used to uncover the genes and signaling pathways affected in wild-type and PDPN-depleted TPC1 and BCPAP cells. We found that changes in the expression of various factors of signaling pathways, like RHOA and RAC1 GTPases and their regulators, are linked with both high PDPN levels and presence of the BRAF V600E mutation. We imply that the suppressed motility of wild-type BCPAP cells results from overactivation of RHOA through natively high PDPN expression. This process is accompanied by inhibition of the PI3K kinase and consequently RAC1, due to overactivation of RAS-mediated signaling and the PTEN regulator.

MiR-576-5p Promotes the Proliferation of Papillary Thyroid Carcinoma through the MAPK4-AKT Pathway.

MicroRNA-576-5p (miR-576-5p) plays an important role in different human cancers. However, the biological function of miR-576-5p in papillary thyroid carcinoma (PTC) is still unclear. In this study, we explored the function and specific role of miR-576-5p in PTC. Expression levels of miR-576-5p in PTC patient tissues and cell lines were determined by reverse transcription-quantitative polymerase chain reaction (qRT‒PCR). Cell counting using cell counting kit-8 (CCK-8), wound healing, and Transwell assays were performed to evaluate the effect of miR-576-5p on the proliferation, migration, and invasion of TPC-1 cells. Expression levels of mitogen-activated protein kinase 4 (MAPK4) and phosphorylation levels of protein kinase B (AKT), extracellular regulated protein kinase (ERK), and P38 mitogen-activated protein kinase (P38) were detected by western blotting or immunohistochemistry (IHC). The expression level of miR-576-5p in PTC tissues and TPC-1 cells was significantly increased. In vitro, overexpression of miR-576-5p promoted the proliferation, migration, and invasion of TPC-1 cells. In addition, MAPK4 was highly expressed in PTC tissues, and miR-576-5p could upregulate the expression of MAPK4. Interestingly, MAPK4 knockdown reversed cell proliferation but not migration and invasion in TPC-1 cells after miR-576-5p was overexpressed. Moreover, overexpression of miR-576-5p induced activation of the AKT pathway in TPC-1 cells, and MAPK4 gene knockout reversed this AKT pathway activation. In this study, we found that miR-576-5p was significantly overexpressed in PTC tissues and TPC-1 cells. In addition, miR-576-5p promoted the proliferation of TPC-1 cells by enhancing expression of MAPK4 and activating the AKT pathway.

Synthesis and Antiproliferative Effect of Halogenated Coumarin Derivatives.

A series of 6- and 6,8-halocoumarin derivatives have been investigated as potential antiproliferative compounds against a panel of tumor and normal cell lines. Cytotoxic effects were determined by the MTT method. To investigate the potential molecular mechanism involved in the cytotoxic effect, apoptosis assay, cell cycle analysis, reactive oxygen species (ROS), and reduced glutathione analysis were performed. Among the screened compounds, coumarins 6,8-dibromo-2-oxo-2H-chromene-3-carbonitrile 2h and 6,8-diiodo-2-oxo-2H-chromene-3-carbonitrile 2k exhibited the most antiproliferative effect in thyroid cancer-derived cells TPC-1. The apoptosis assay showed that both 2h and 2k induced apoptosis in TPC-1 thyroid cancer cells. According to these experiments, both coumarins induced a slight increase in TPC-1 cells in the G2/M phase and a decrease in the S phase. A significant increase in ROS levels was observed in TPC-1 treated with diiodocoumarin 2k, while the dibromocoumarin 2h induced a decrease in ROS in a dose and time-dependent manner.

DACH1 inhibits the proliferation and migration of papillary thyroid carcinoma.

DACH1 is an important component of the retinal determinate gene network (RDGN), which regulates the expression of target genes by directly binding or interacting with other factors. DACH1 shows inhibitory effects in most tumors, but its role in papillary thyroid carcinoma is unclear and warrants further investigation. We assessed the expression of DACH1 in different tissues and correlation with immune infiltration by The Cancer Genome Atlas (TCGA) and Tumor Immune Estimation Resource (TIMMER2.0 databases). The effects of DACH1 on the proliferation and migration of TPC-1 and Bcpap cells were assessed by cell viability assay, colony formation assay, wound healing assay, transwell migration assay, and flow cytometry. Finally, the effects of DACH1 on CXCL8, CXCL10, and CXCL12 expression in Nthy-ori-3-1, TPC-1 and Bcpap cells were assessed by enzyme-linked immunosorbent assay kit and real-time polymerase chain reaction, respectively. The results showed that DACH1 was differentially expressed in different tumors and tissues. Basal expression of DACH1 was lower in thyroid and papillary thyroid carcinoma than in other normal tissues and corresponding tumors, and positively correlated with CD8+ T cell infiltration. In Nthy-ori-3-1, TPC-1 and Bcpap cells, overexpression of DACH1 inhibitedcell migration and proliferation, and the opposite results was obtained by knocking down DACH1 using small interfering RNA. We also demonstrated that DACH1 regulated chemokines CXCL8, CXCL10, and CXCL12, thereby modulating tumor immunity.

ODP272 ROUNDUP IS AN IMPORTANT DISRUPTOR THAT CAUSES PROLIFERATIVE EFFECTS ON NORMAL AND ON CANCER THYROID CELLS

Abstract Glyphosate has long be suspected to promote endocrine disruptive and even carcinogenic effects, but little is known about its effect at different concentrations on normal and on cancer thyroid cells. Since the endocrine system responds to low chemical concentrations and abnormal cells may have different sensitivity, it is essential to determine the effects of this compound at different levels, including the Acceptable Occupational Exposure Level (AOEL) and the Acceptable Daily Intake (IDA) doses. We used two human thyroid-derived cell lines strains, Nthy-ori 3-1 (from thyroid normal follicular cells) and TPC-1 (from papillary carcinoma) to test 15 different concentrations of Roundup® Original DI, ranging from 6.5 µg/L to 6.500 µg/L, including AOEL and IDA according to the Technical Note Process No. 25351. 056754/2013-17 of the National Health Surveillance Agency (ANVISA) in Brazil. Trypan blue assay and Cell Counting Kit – 8 (CCK-8) were employed to evaluate cell viability and cytotoxicity. Exposures were made for 24 and 48 hours in technical and biological triplicates. We observed a decrease in the number of viable cells at the 24h exposure compared to 48h in both strains. Exposure of Nthy-ori 3-1 and TPC-1 cells for 24h at the AOEL concentration (160µg/L) caused death of 52% and 58% of the cells, respectively, whereas IDA (830µg/L) resulted in 43% e 58% cell death. These data evidence an important disruptive effect of Roundup Original DI® at the acceptable concentrations. After 48h of exposure, AOEL and IDA concentrations caused a lower number of Nthy-ori 3-1 (19% e 15%) and TPC-1 (29% e 23%) dead cells, respectively, suggesting both thyroid strains have compensatory mechanisms that protect them from longer exposures to the herbicide. The CCK-8 assay, on the contrary, showed a proliferative effect on small concentrations: 6.5 µg/L increased the number of viable Nthy-ori 3-1 and TPC-1 cells to 113% and 118%, respectively, at 24h, and this stimulatory effect persisted after 48h of exposure. In contrast, AOEL and IDA concentrations produced a reduction in the number of both strains cells, likewise observed with the Trypan blue assay. Our data demonstrate that thyroid cells exposed to Roundup Original DI® present a non-monotone dose-response curve, which is a well-known characteristic of an endocrine disruptor. The proliferative effect produced on thyroid cells, especially TPC-1 mutant cells, by very low concentrations of this glyphosate-based herbicide widely used around the world, suggests that it may play a role in the increasing incidence rate of thyroid nodules and/or cancer that have been observed worldwide. Presentation: No date and time listed

ODP276 THYROID CANCER CELLS ARE MORE RESISTANT THAN NORMAL CELLS TO BISPHENOL-A CYTOTOXIC EFFECT

Abstract Bisphenol-A (BPA) is a well-known endocrine disruptor that produces important effects on thyroid metabolism, can interfere with TSH transcriptional activity, alter the transcription of T3-associated genes and, thus, modulate its activity in different cell lines. However, its direct effects on thyroid cells, especially on mutated thyroid cells, are still poorly understood. We tested 14 different concentrations of BPA, ranging from 0.4µg/mL to 400µg/mL, including LME (Specific Migration Limit =1μg/mL, recommended as acceptable by the Brazilian National Health Surveillance Agency-ANVISA) on two human thyroid cell lines: Nthy-ori 3-1 (from normal thyroid follicular cells) and TPC-1 (from papillary carcinoma). Cell viability analysis was made using the Trypan blue assay on cells exposed to BPA for 24h and 48h in technical and biological triplicate. We observed a non-monotone dose-response curve, characteristic of disruptors’ effect, on both cell lines, with greater cytotoxic effect with higher doses. In fact,100μg/mL already killed almost all Nthy-ori 3-1 cells (99% at 24h and 97% at 48h), whereasthe doses of 0.78µg/mL; 4µg/mL and 12.5µg/mL caused 50% kill. Optical microscopy revealed some irregular cells lost on the plate, and few debris with50μg/mL. Concentrations of 100 to 400µg/mL produced many dark small cells detached from the plate and clumps of dead cells. The SCI dose (1µg/mL - allowed in daily life), resulted in 79% of Nthy-ori 3-1 cell death after 24h of exposure. However, only 7% of the cell were dead after 48h of exposure to the SCI dose. This reduction in death rate after longer exposure to BPA was observed at all concentrations, suggesting a potent cellular recovery mechanism. The TPC-1 strain showed a different behavior, with a much more linear dose-dependent cytotoxic effect and much more resistance to the harmful effects of BPA at low doses. In fact, 0.4μg/mL to 3.125μg/mL produced less than 10% cell death, and microscopy confirmed the maintenance of the integrity of virtually all cells. On the contrary, BPA concentrations from 100 to 400µg/mL killed almost all TPC-1 cells at both 24h and 48h times. We conclude that this thyroid papillary carcinoma cell line is more resistant than the normal thyroid lineage to low BPA concentrations (from 0.4 to 50µg/mL) but this endocrine disruptor is highly cytotoxic at higher concentrations, causing more than 90% cell deaths on both normal and mutated thyroid cell lines. Presentation: No date and time listed

RF11 | PSAT369 CDK7 and CDK9 Inhibition Induces RNA Polymerase II Ubiquitination and Proteasome Degradation in Thyroid Cancer Cells

Abstract "Superhenhanced" transcription of specific oncogenes by aberrant looping of upstream enhancer elements to marked transcriptional regulatory regions is a mechanism of oncogene overexpression. Therefore, compounds that target key positive regulators of RNA polymerase II (RNAPII) activity, such as CDK7 and CDK9, can relatively selectively reduce expression of such superenhanced oncogenes and are in clinical trials. We have previously reported activity of the CDK7 inhibitor THZ1 in medullary thyroid cancer (MTC) along with identification of a superenhancer in RET. CDK7 inhibitors also are reported to be active in anaplastic thyroid cancer cell lines. In the present study, we extend this work to both CDK7 and CDK9 inhibitors, and to a more extensive set of thyroid cancer cell lines. We selected 8 thyroid cancer cell lines (TPC1, FTC133, BCPAP, SW1376; K1, THJ-16T, C643, and 8505C) from a variety of histological subtypes with different driver mutations for treatment with a CDK7 (THZ1) or CDK9 (AZD4573) inhibitors. Cell viability assays were performed to determine IC50 and Western blots were performed for target validation. IC50 values ranged from 5-100 nM for both compounds for all cell lines demonstrating broad activity and robust activity. Unexpectedly, both compounds inhibited both CDK7 RNAPII phosphorylation (Ser5) and CDK9 RNAPII phosphorylation (Ser2) due to a loss of total RNAPII protein levels in all cell lines. Further time course studies two cell lines, 8505C (BRAFV600E) and TPC1 (RET/PTC) showed the loss off RNAPII occurred rapidly (1-3 hours) with stable RNAPII mRNA levels by qRTPCR. We therefore hypothesized that RNAPII stalling induced by inhibition of CDK7 may cause loss of RNAPII protein stability through ubiquitination and proteasome-mediated degradation. Immunoprecipitation-Western blots demonstrated variable baseline levels of RNAPII ubiquitination in 8505C (low) and TPC1 (high) cells. THZ1 treatment induced ubiquitination of RNAPII in 8505C cells signficantly, while a modest increase beyond the higher baseline occurred in TPC1. Bortezomib (proteasome inhibitor) treatment rescued THZ1-reduced RNAPII protein levels in both cell lines. In summary, human thyroid cancer cell lines are broadly sensitive to CDK7 and CDK9 inhibitors. CDK 7 and 9 inhibitors reduce RNAPII levels and THZ1 induces RNAPII ubiquitination and proteasome degradation demonstrating a new mode of action for the reduced RNAPII activity induced by these inhibitors. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Saturday, June 11, 2022 1:42 p.m. - 1:47 p.m.

Gene Analysis of Cancer Stem Cells in Thyroid Cancer

<i>Objective </i>Recurrence and drug resistance in different thyroid cancer in the clinic are associated with tumor stem cells. To investigate the differences in transcript levels of tumor stem cell-associated genes in different thyroid cancer cell lines and their correlation with tumor pathways for identifing factors affecting thyroid cancer progression. <i>Methods </i>Wound healing assay was performed to analyze cell migration; to compare the transcript levels of tumor stem cell related genes CD133, OCT4, ABCG2, ALDH2, Nanog, CD44, CD24, EMT and thyroid specific genes in different thyroid cancer cell lines; to analyze the correlation between the above genes and tumor pathways and the correlation between genes using R software GSVA package combined with TCGA database. <i>Results</i> Wound healing results showed that thyroid cancer cell lines TPC-1 and ATC healed faster than normal cells. qPCR showed significantly different expression of EGFR, CD44, CD24 and ABCG2 in ATC and TPC-1 compared to Nthy-ori 3-1. EGFR and ABCG2 were highly expressed in ATC cell lines, while CD24 gene was highly expressed in TPC -1; TTF2 was significantly low expressed in both ATC and TPC-1 cell lines. Correlation analysis showed that ABCG2 and OCT4 were negatively correlated with inflammatory response, ALDH2 with P53 signaling pathway, CD24 with ECM degradation and P53 signaling pathway, Nanog with MYC target gene pathway, and CD44 with reactive oxygen species (ROS) gene upregulation in thyroid cancer; EGFR was positively correlated with TGFB signaling pathway. The genes ALDH2, ABCG2 and CD44 were significantly and positively correlated with TPO and TG. <i>Conclusion </i>The transcriptional levels of tumor stem cell genes differed greatly among different types of thyroid cancer and were significantly associated with tumor signaling pathways. It may be related to signaling pathways that exacerbate the development of thyroid cancer.

CircNDST1 promotes papillary thyroid cancer progression via its interaction with CSNK2A1 to activate the PI3K–Akt pathway and epithelial–mesenchymal transition

BackgroundMultiple studies have established a strong relationship between circRNA and cancer progression. Cervical lymph node metastasis is a key factor influencing the surgical approach and distant metastasis of papillary thyroid cancer (PTC). However, the role of circNDST1 in PTC has not been investigated. Our research focused on revealing the function and mechanism of action of circNDST1 in PTC.MethodsHigh-throughput sequencing and qPCR were used to assess the expression of circRNA in PTC tissues with extensive cervical lymph node metastasis and circNDST1 in cell lines, respectively. The proliferative effects of circNDST1 in vitro and in vivo were analyzed using CCK8, clone formation assay, EdU, and nude mouse tumorigenesis assay. The transwell scratch assay was employed in the scrutiny of the effect of circNDST1 on the migration and invasion abilities of thyroid cancer cells, while circNDST1’s influence on the PI3K–Akt pathway and the Epithelial–Mesenchymal Transition (EMT) key protein expression was evaluated utilizing RNA sequencing and western blot. RNA pull-down and RIP were used to examine the binding of circNDST1 to CSNK2A1.ResultsCircNDST1 was highly expressed in PTC cell lines, but knocking it down inhibited the proliferation, migration, and invasive abilities of TPC1 and KTC1 cell lines. CircNDST1 bonded with CSNK2A1 and promoted the interaction between CSNK2A1 and Akt, leading to the activation of the PI3K–Akt pathway and EMT.ConclusionCircNDST1’s high expression boosted thyroid cancer progression through the activation of the PI3K–Akt pathway and EMT in a CSNK2A1-dependent manner

Influence of Dehydroxymethylepoxyquinomicin on Radiosensitivity of Thyroid Carcinoma TPC-1 Cells.

Objective To investigate the influence of dehydroxymethylepoxyquinomicin (DHMEQ), an NF-κB inhibitor, on radiosensitivity of thyroid carcinoma (TC) TPC-1 cells. Methods The isolation of CDl33 positive cells (CD133+ TPC-1) and negative cells (CD133− TPC-1) from TPC-1 cells used immunomagnetic bead sorting. After verification of the toxicity of DHMEQ to cells by MTT and cell cloning assays, the cells were divided into four groups, of which three groups were intervened by DHMEQ, 131I radiation, and DHMEQ +131I radiation, respectively, while the fourth group was used as a control without treatment. Alterations in cell growth, apoptosis, and cell cycle were observed. Results DHMEQ had certain toxic effects on TPC-1 cells, with an IC50 of 38.57 μg/mL (P < 0.05). DHMEQ inhibited CD133+ and CD133- TPC-1 proliferation and their clonogenesis after irradiation. DHMEQ + radiation contributed to a growth inhibition rate and an apoptosis rate higher than either or them alone (P < 0.05), with a more significant effect on CD133− TPC-1 than CD133+ TPC-1 under the same treatment conditions (P < 0.05). Conclusion DHEMQ can increase the radiosensitivity of TC cells to 131I, inhibit tumor cell growth, and promote apoptosis. However, its effect is less significant on CD133+ TPC-1 compared with CD133− TPC-1, which may be related to the stem cell-like properties of CD133+ cells. In the future, the application of DHMEQ in TC 131I radiotherapy will effectively improve the clinical effect of patients.

Sinomenine Hydrochloride Promotes TSHR-Dependent Redifferentiation in Papillary Thyroid Cancer.

Radioactive iodine (RAI) plays an important role in the diagnosis and treatment of papillary thyroid cancer (PTC). The curative effects of RAI therapy are not only related to radiosensitivity but also closely related to the accumulation of radionuclides in the lesion in PTC. Sinomenine hydrochloride (SH) can suppress tumor growth and increase radiosensitivity in several tumor cells, including PTC. The aim of this research was to investigate the therapeutic potential of SH on PTC cell redifferentiation. In this study, we treated BCPAP and TPC-1 cells with SH and tested the expression of thyroid differentiation-related genes. RAI uptake caused by SH-pretreatment was also evaluated. The results indicate that 4 mM SH significantly inhibited proliferation and increased the expression of the thyroid iodine-handling gene compared with the control group (p < 0.005), including the sodium/iodide symporter (NIS). Furthermore, SH also upregulated the membrane localization of NIS and RAI uptake. We further verified that upregulation of NIS was associated with the activation of the thyroid-stimulating hormone receptor (TSHR)/cyclic adenosine monophosphate (cAMP) signaling pathway. In conclusion, SH can inhibit proliferation, induce apoptosis, promote redifferentiation, and then increase the efficacy of RAI therapy in PTC cells. Thus, our results suggest that SH could be useful as an adjuvant therapy in combination with RAI therapy in PTC.