Anaplastic Thyroid Cancer Cell Proliferation Research Articles

Macrophage-Induced Carboxypeptidase A4 Promotes the Progression of Anaplastic Thyroid Cancer.

Background: The density of tumor-associated macrophages in the tumor microenvironment of anaplastic thyroid cancer (ATC) is associated with poor prognosis. However, the crosstalk between macrophages and ATC cells is poorly understood. This study aimed to examine the impact of macrophages on cancer cell phenotypes. We found a new mediator between M2 macrophages and ATC cells through proteomics analysis. Methods: The role of macrophages in proliferation, migration, and invasion of ATC cells was evaluated using coculture assay and conditioned medium (CM). Secretory factors in the CM from single or coculture were identified using liquid chromatography-tandem mass spectrometry proteomics analysis. We evaluated the role of the secretory factor in proliferation, migration, and invasion of cancer cells. In vivo xenograft model was used to evaluate the effect of the factor. Results: M2 macrophages significantly increased the proliferation, migration, and invasion of ATC cells, whereas M1 macrophages decreased the proliferation, migration, and invasion of ATC cells. Based on proteomic analysis of CM, we identify carboxypeptidase A4 (CPA4) as a mediator of the crosstalk between macrophages and ATC cells. CPA4 was only detected in the coculture media of M2 macrophage/8505C, and its expression in cancer cells increased by M2 macrophage. The expression of CPA4 protein was significantly higher in human thyroid cancers, particularly in ATCs, than normal and benign tissues. A bioinformatics analysis of public data revealed that CPA4 expression was associated with poor prognosis and dedifferentiation of thyroid cancer. Knockdown of CPA4 suppressed proliferation, colony formation, migration, and invasion of ATC cells, consistent with the decrease of STAT3, ERK, and AKT/mTOR phosphorylation and epithelial-mesenchymal transition (EMT) marker expression. In addition, the increased expression of CPA4 in cancer cells by M2 macrophage stimulation induced the polarization of macrophages to the M2 phenotype, which formed a positive feedback loop. Xenograft tumors did not develop after CPA4 knockdown. Conclusions: Our data suggest that CPA4 stimulates the progression of thyroid cancer by mediating between M2 macrophages and ATC cells. CPA4 can be a new therapeutic target for the treatment of patients with ATC.

SIRT7 promotes the proliferation and migration of anaplastic thyroid cancer cells by regulating the desuccinylation of KIF23

ObjectiveThis study was designed to investigate the regulatory effects of kinesin family member (KIF) 23 on anaplastic thyroid cancer (ATC) cell viability and migration and the underlying mechanism.MethodsReverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to analyze the levels of KIF23 in ATC cells. Besides, the effects of KIF23 and sirtuin (SIRT) 7 on the viability and migration of ATC cells were detected using cell counting kit-8, transwell and wound healing assays. The interaction between SIRT7 and KIF23 was evaluated by co-immunoprecipitation (Co-IP) assay. The succinylation (succ) of KIF23 was analyzed by western blot.ResultsThe KIF23 expression was upregulated in ATC cells. Silencing of KIF23 suppressed the viability and migration of 8505C and BCPAP cells. The KIF23-succ level was decreased in ATC cells. SIRT7 interacted with KIF23 to inhibit the succinylation of KIF23 at K537 site in human embryonic kidney (HEK)-293T cells. Overexpression of SIRT7 enhanced the protein stability of KIF23 in HEK-293T cells. Besides, overexpression of KIF23 promoted the viability and migration of 8505C and BCPAP cells, which was partly blocked by silenced SIRT7.ConclusionsSIRT7 promoted the proliferation and migration of ATC cells by regulating the desuccinylation of KIF23.

Synergy between isobavachalcone and doxorubicin suppressed the progression of anaplastic thyroid cancer through ferroptosis activation.

The objective of this study was to explore the effects and mechanisms of the combination of isobavachalcone (IBC) and doxorubicin (DOX) on the progression of anaplastic thyroid cancer (ATC). Cell viability of 8505C and CAL62 cells was observed by CCK-8 assay. Kits were used to detect the presence of reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), and cellular iron. Protein expression of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) was detected using western blot, and CD31 was detected through immunofluorescence. Tumor xenograft models of 8505C cells were constructed to observe the effect of IBC and DOX on ATC growth in vivo. The co-administration of IBC and DOX exhibited a synergistic effect of suppressing the growth of 8505C and CAL62 cells. The concurrent use of IBC and DOX resulted in elevated iron, ROS, and MDA levels, while reducing GSH levels and protein expression of SLC7A11 and GPX4. However, the Fer-1 ferroptosis inhibitor effectively counteracted this effect. In vitro and in vivo, the inhibitory effect on ATC cell proliferation and tumor growth was significantly enhanced by the combination of IBC and DOX. The combination of IBC and DOX can inhibit the growth of ATC by activating ferroptosis, and might prove to be a potent chemotherapy protocol for addressing ATC.

Thyroid hormone receptor α1: a novel regulator of thyroid cancer cell differentiation.

Thyroid hormone receptor α1 (TRα1) mediates the genomic actions of thyroid hormone (T3). The biology of TRα1 in growth and development has been well studied, but the functional role of TRα1 in cancers remains to be elucidated. Analysis of the human thyroid cancer database of The Cancer Genome Atlas (TCGA) showed that THRA gene expression is lost in highly dedifferentiated anaplastic thyroid cancer (ATC). We, therefore, explored the effects of TRα1 on the progression of ATC. We stably expressed TRα1 in two human ATC cell lines, THJ-11T (11T-TRα1 #2, #7, and #8) and THJ-16T (16T-TRα1 #3, #4, and #8) cells. We found that the expressed TRα1 inhibited ATC cell proliferation and induced apoptosis. TCGA data showed that THRA gene expression was best correlated with the paired box gene 8 (PAX8). Consistently, we found that the PAX8 expression was barely detectable in parental 11T and 16T cells. However, PAX8 gene expression was elevated in 11T- and 16T-TRα1-expressing cells at the mRNA and protein levels. Using various molecular analyses, we found that TRα1 directly regulated the expression of the PAX8 gene. Single-cell transcriptomic analyses (scRNA-seq) demonstrated that TRα1 functions as a transcription factor through multiple signaling pathways to suppress tumor growth. Importantly, scRNA-seq analysis showed that TRα1-induced PAX8, via its transcription program, shifts the cell landscape of ATC toward a differentiated state. The present studies suggest that TRα1 is a newly identified regulator of thyroid differentiation and could be considered as a potential therapeutic target to improve the outcome of ATC patients.

Type 2 deiodinase is expressed in anaplastic thyroid carcinoma and its inhibition causes cell senescence.

Anaplastic thyroid cancer (ATC) is a rare thyroid tumor that frequently originates from the dedifferentiation of a well-differentiated papillary or follicular thyroid cancer. Type 2 deiodinase (D2), responsible for the activation of the thyroid hormone thyroxine into tri-iodothyronine (T3), is expressed in normal thyroid cells and its expression is strongly downregulated in papillary thyroid cancer. In skin cancer, D2 has been associated with cancer progression, dedifferentiation, and epithelial-mesenchymal transition. Here, we show that D2 is highly expressed in anaplastic compared to papillary thyroid cancer cell lines and that D2-derived T3 is required for ATC cell proliferation. D2 inhibition is associated with G1 growth arrest and induction of cell senescence, together with reduced cell migration and invasive potential. Finally, we found that mutated p5372R(R248W), frequently found in ATC, is able to induce D2 expression in transfected papillary thyroid cancer cells. Our results show that the action of D2 is crucial for ATC proliferation and invasiveness, providing a potential new therapeutic target for the treatment of ATC.

Ellagic acid inhibits cell proliferation, migration, and invasion of anaplastic thyroid cancer cells via the Wnt/β-catenin and PI3K/Akt pathways.

Anaplastic thyroid cancer (ATC) is a rare but lethal human malignant cancer with no known cure. Ellagic acid (EA), a natural plant extract, has shown antitumor activity against multiple cancers; however, its effects on the malignant phenotypes of ATC cells remain unknown. This study aimed to evaluate the effects of EA on proliferation, migration, and invasion of ATC cells and further explore the associated signaling mechanisms. The normal human thyroid cell line Nthy-ori3-1 and ATC cell line BHT-101 were used. Cytotoxicity assay was performed using the Cell Counting kit-8 (CCK-8) assay. Cell proliferation, migration, and invasion assays were performed using the CCK-8 and colony formation, wound healing, and Transwell invasion assays, respectively. Western blotting was used to detect the levels of related proteins. β-catenin nuclear protein levels were measured to evaluate the Wnt/β-catenin pathway. The phosphorylation level of the Akt protein was measured and calculated to evaluate the PI3K/Akt pathway. LiCl and IGF-1 were used as pathway agonists to determine the involvement of the corresponding pathway. The results showed that EA inhibited the proliferation, migration, and invasion of ATC cells. Furthermore, both the Wnt/β-catenin and PI3K/Akt pathways were suppressed by EA treatment, and activation of these two pathways reversed the EA-induced inhibition of the pathological phenotypes of ATC cells. These findings demonstrate that EA inhibits proliferation, migration, and invasion of ATC cells by suppressing the Wnt/β-catenin and PI3K/Akt pathways, suggesting that EA is a potential drug candidate for treating ATC and provides a theoretical basis for further in vivo experiments and clinical applications.

Blocking CDK7-Mediated NOTCH1-cMYC Signaling Attenuates Cancer Stem Cell Activity in Anaplastic Thyroid Cancer.

Background: Anaplastic thyroid cancer (ATC) is an aggressive solid cancer in humans with few treatment options. Recent studies suggest that aberrant gene transcription could contribute to aggressive ATC progression. To test this hypothesis, we assessed if blocking cyclin-dependent protein 7 (CDK7) activity could impede ATC progression through attenuation of cancer stem cell (CSC) activity. Methods: We treated cell lines isolated from human ATC (THJ-11T and -16T) and xenograft mice induced by these cells with the CDK7 inhibitor THZ1. Through integrative transcriptome analyses we found that the NOTCH1-cMYC signaling axis was a potential target of CDK7 inhibition in ATC. To determine the regulatory action of NOTCH1-cMYC signaling in CSC maintenance, we evaluated the effect of a selective NOTCH1 inhibitor, crenigacestat, on CSC capacities in ATC. Results: THZ1 markedly inhibited proliferation of ATC cells and xenograft tumor growth by blocking cell cycle progression and inducing apoptosis. NOTCH1 was sensitive to suppressive transcription mediated by CDK7 inhibition and was highly enriched in tumorspheres from ATC cells. Treatment of ATC cells with either crenigacestat or THZ1 blocked formation of tumorspheres, decreased aldehyde dehydrogenase activity, and suppressed in vivo initiation and growth of tumors induced by ATC cells, indicating that NOTCH1 was a critical regulator of CSC activity in ATC. Furthermore, we demonstrated that cMYC was a downstream target of NOTCH1 signaling that collaboratively maintained CSC activity in ATC. Of note, genomic analysis showed that low CDK7 expression contributed to longer disease-free survival of thyroid cancer patients. Conclusions: NOTCH1 is a newly identified CSC regulator. Targeting NOTCH1-cMYC signaling is a promising therapeutic strategy for ATC.

Effect of Lactate Export Inhibition on Anaplastic Thyroid Cancer Growth and Metabolism.

Anaplastic thyroid cancer (ATC) is an aggressive malignancy without effective treatments. ATC cells demonstrate upregulated glycolysis (Warburg effect), generating lactate that is subsequently exported by monocarboxylate transporter 4 (MCT4). This study aims to determine whether MCT4 inhibition can suppress ATC growth. ATC cell lines 8505C, JL30, and TCO1 were grown in low (3 mmol/L; LG) or high (25 mmol/L; HG) glucose medium containing the lactate shuttle inhibitors acriflavine (10-25 μmol/L; ACF), syrosingopine (100 µmol/L; SYR), or AZD3965 (20 µmol/L; AZD). Lactate level and cell proliferation were measured with standard assays. Seahorse analysis was performed to determine glycolytic response. Compared with HG, addition of ACF to LG decreased lactate secretion for both 8505C (p < 10-5) and JL30 (p < 10-4) cells, whereas proliferation was also reduced (p < 10-4 and 10-5, respectively). During Seahorse analysis, addition of oligomycin increased acidification by 84 mpH/min in HG vs 10 mpH/min in LG containing ACF (p < 10-5). Treatment with LG and SYR drastically diminished 8505C and TCO1 growth vs HG (p < 0.01 for both). LG and AZD treatment also led to reduced proliferation in tested cell lines (p ≤ 0.01 for all) that was further decreased by addition of ACF (p < 10-4 vs HG, p ≤ 0.01 vs LG and AZD). Inhibition of lactate shuttles significantly reduced proliferation and glycolytic capacity of ATC cells in a low-glucose environment. Targeting suppression of glycolytic and lactate processing pathways may represent an effective treatment strategy for ATC.

USP26 promotes anaplastic thyroid cancer progression by stabilizing TAZ

Anaplastic thyroid cancer (ATC) is one of the most lethal and aggressive human malignancies, with no effective treatment currently available. The Hippo tumor suppressor pathway is highly conserved in mammals and plays an important role in carcinogenesis. TAZ is one of major key effectors of the Hippo pathway. However, the mechanism supporting abnormal TAZ expression in ATC remains to be characterized. In the present study, we identified USP26, a DUB enzyme in the ubiquitin-specific proteases family, as a bona fide deubiquitylase of TAZ in ATC. USP26 was shown to interact with, deubiquitylate, and stabilize TAZ in a deubiquitylation activity-dependent manner. USP26 depletion significantly decreased ATC cell proliferation, migration, and invasion. The effects induced by USP26 depletion could be rescued by further TAZ overexpression. Depletion of USP26 decreased the TAZ protein level and the expression of TAZ/TEAD target genes in ATC, including CTGF, ANKRD1, and CYR61. In general, our findings establish a previously undocumented catalytic role for USP26 as a deubiquitinating enzyme of TAZ and provides a possible target for the therapy of ATC.

Expression of colorectal neoplasia differentially expressed in anaplastic thyroid carcinoma and its effect on cancer cell proliferation.

BackgroundThe incidence of anaplastic thyroid cancer (ATC) is high among human cancers. Colorectal neoplasia differentially expressed (CRNDE) is highly expressed in common tumors, and is therefore a potential molecular target for anti-tumor therapy. However, the function of CRNDE in ATC remains elusive.MethodsThe Gene Expression Omnibus (GEO) database was used to screen the differential expression of long-noncoding RNA (lncRNA) in ATC tissues. The Cancer Genome Atlas (TCGA) database was used to analyze the expression of CRNDE in thyroid cancer (THCA) tissues and its impact on patient prognosis. Quantitative real-time PCR (qRT-PCR) was used to determine the expression level of CRNDE in tumor and control tissues. The biological function of CRNDE in THCA was explored using TCGA RNA sequencing (RNA-seq) data analysis. ATC cell lines with low and high CRNDE expression were selected for CRNDE siRNA transfection, and the proliferation of cells was detected in each group.ResultsThe GEO and TCGA databases analysis results showed that CRNDE was highly expressed in ATC tissues, which is related to the poor prognosis of THCA patients. Also, the expression of CRNDE in the ATC cell line, ARO (human thyroid cancer cell line), was relatively high, while the expression in sw579 is relatively low. Therefore, ARO and sw579 were chosen for CRNDE small interfering RNA (siRNA) transfection. Compared with negative control (si-NC), the expression of CRNDE in si-CRNDE-1, si-CRNDE-2, and si-CRNDE-3 was reduced, indicating that the inhibitory effect was significantly enhanced and the cell proliferation ability was reduced, and the cell cycle is arrested in the G0/G1 phase. Finally, it was found that the wnt3a, β-catenin, and cyclinD1 protein expressions of si-CRNDE-1 and si-CRNDE-2 were significantly reduced.ConclusionsThe high expression of CRNDE in ATC tissues may promote the proliferation of ATC cells by regulating the Wnt/β-catenin signaling pathway. CRNDE may be a potential molecular target for the treatment of ATC.

ASPM Promotes the Progression of Anaplastic Thyroid Carcinomas by Regulating the Wnt/β-Catenin Signaling Pathway.

Background Abnormal spindle-like microcephaly-associated protein (ASPM) is closely correlated with several malignant tumors, whereas little is known about the role of ASPM in anaplastic thyroid cancer (ATC). Herein, we sought to investigate whether ASPM is involved in the pathogenesis of ATC and the underlying mechanisms. Methods The data from two data sets (GSE76039 and GSE33630) were extracted and analyzed for the expression of ASPM, followed by a further validation in collected ATC patients using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The effect of ASPM on cell proliferation, migration, invasion, and cell cycle was explored in ATC cell lines by in vitro inhibition of ASPM, while ASPM-mediated tumorigenicity was investigated in a xenograft tumor model. The involvement of Wnt/β-catenin signaling pathway was also investigated. Results ASPM was overexpressed in ATC patients and cell lines. In vitro knockdown of ASPM inhibited the proliferation, migration, and invasion capabilities of ATC cells and induced cell cycle arrest. Wnt/β-catenin signaling was suppressed in response to ASPM inhibition, while rescue of β-catenin expression restored the impaired biological functions of ATC cells. In vivo transplantation of ASPM-knockdown cells inhibited the growth of tumors. Conclusions Upregulation of ASPM promotes the malignant properties of ATC cells and contributes to tumorigenesis through the Wnt/β-catenin signaling pathway.

SOX13/TRIM11/YAP axis promotes the proliferation, migration and chemoresistance of anaplastic thyroid cancer.

Anaplastic thyroid cancer (ATC) is one of the most aggressive and virulent solid tumors. The ubiquitin proteasome system presents in all eukaryotic cells and is essential for cellular homeostasis. While its underlying role in ATC remains largely unclear. TRIM11 is an E3 ubiquitin ligase and has been reported to act as an oncogene in several human cancers. The present study aims to reveal the oncogenic function of TRIM11 in ATC. Western blot was used to measure the protein expression of TRIM11 and YAP, while the YAP target genes were measured by real-time PCR. CCK8 assay was used to detect cell viability; wound-healing assay and transwell assay were used to measure the migration ability of ATC. The xeno-graft tumor model was used for in vivo study. Immuno-precipitation assay was used to detect the interaction domain between YAP and TRIM11. And the ubiquitin-based Immuno-precipitation assays were used to detect the specific ubiquitination manner happened on YAP. TRIM11 depletion significantly decreases cell proliferation and migration capabilities of ATC cells, and elevates cell sensitivity to chemotherapy, which effect could be further rescued by YAP overexpression. TRIM11 depletion decreases YAP protein level and YAP/TEAD target genes, such as CTGF, ANKRD1 and CYR61 in ATC. Indicating that TRIM11 is a regulator of Hippo signaling pathway. Immuno-precipitation assay shows that the RING domain of TRIM11 is essential for the interaction with WW domain of YAP. Further mechanistic analysis suggests that TRIM11 promotes the mono-ubiquitination of YAP, thus prolongs its protein half. Furthermore, TRIM11 promoter analysis revealed that SOX13 activates TRIM11 transcription by binding to the promoter of TRIM11. In summary, our study describes the oncogenic function of TRIM11 in ATC, which acts as a post-translational modulating factor of Hippo pathway. Targeting TRIM11 may be a potential therapeutic method for ATC treatment.

Dsg2-mediated c-Met activation in anaplastic thyroid cancer motility and invasion.

Anaplastic thyroid cancer (ATC) is a rapidly growing, highly metastatic cancer with limited therapeutic alternatives, thus targeted therapies need to be developed. This study aimed to examine desmoglein 2 (Dsg2) expression in ATC and its biological role and potential as a therapeutic target in ATC. Consequently, Dsg2 was downregulated or aberrantly expressed in ATC tissues. ATC patients with low Dsg2 expression levels also presented with distant metastasis. Dsg2 depletion significantly increased cell migration and invasion, with a relatively limited effect on ATC cell proliferation in vitro and increased distant metastasis in vivo. Dsg2 knockdown induced cell motility through the hepatocyte growth factor receptor (HGFR, c-Met)/Src/Rac1 signaling axis, with no alterations in the expression of EMT-related molecules. Further, specific targeting of c-Met significantly inhibited the motility of shDsg2-depleted ATC cells. Decreased membrane Dsg2 expression increased the metastatic potential of ATC cells. These results indicate that Dsg2 plays an important role in ATC cell migration and invasiveness. Therapies targeting c-Met might be effective among ATC patients with low membrane Dsg2 expression levels, indicating that the analysis of Dsg2 expression potentially provides novel insights into treatment strategies for ATC.

Abstract 1838: Accumulation of DNA:RNA hybrids (R-loops) and cell death after YM155 treatment of anaplastic thyroid cancer hints at a novel therapeutic approach

Abstract Introduction: Anaplastic thyroid cancer (ATC) is a rapidly fatal disease with a median survival of less than 6 months. Although rare, ATC is responsible for a disproportionate number of thyroid cancer deaths and is highly resistant to common cancer therapies. YM155 is a small molecule drug identified in a screen by the National Cancer Institute that potently kills ATC cells in vitro and in vivo. We have previously shown the ability of YM155 to inhibit ATC cell proliferation, however, the mechanism is still unclear. This study investigates the DNA damage and accumulation of R-loops that is seen with YM155 treatment of ATC cells. R-loops are structures characterized by a DNA:RNA hybrid with a displaced single strand of DNA and can be formed by transcription-replication collision, nucleotide depletion, and other situations where transcription is stalled. Materials &amp; Methods: Clonogenic assays were performed in four ATC cell lines (ACT1, THJ11T, THJ16T, and THJ29T) to study the effect of YM155 on cell survival. DNA damage was measured by COMET assay and by counting foci of phosphorylated histone H2AX (γH2AX). R-loops were measured via immunofluorescent microscopy with S9.6 anti-R-loop antibody and anti-nucleolin antibody in conjunction with an AI algorithm using inFormTM cell segmentation software to quantify non-nucleolar nuclear R-loop signal. Results: We show in multiple ATC cell lines that YM155 potently kills ATC cells at low nanomolar concentrations. COMET assay found significant increases in DNA damage after treatment with 10 nM YM155, with large increases in tail moment seen by 24 hours in three ATC cell lines (p &amp;lt 0.001 for all) with no change in primary benign thyroid (BT) cells. Similar trends were seen when measuring formation of γH2AX foci, a marker for double strand DNA breaks. R-loop immunofluorescence more than doubled with 10 nM YM155 treatment (p &amp;lt 0.001) similar to hydroxyurea, a positive control. This finding was confirmed when addition of RNAseH, which is specific for RNA in DNA:RNA hybrids, reduced R-loop signal down to baseline levels. Conclusion: YM155's ability to kill ATC cells at low concentrations while sparing BT cells coincides with increases in DNA damage and accumulation of R-loops. A variety of factors aid in the resolution of R-loops, and we have previously shown patient data exhibiting overexpression of DNA topoisomerase genes TOP1 and TOP2A. Combined with published information describing the ability of YM155 to inhibit Topoisomerase Iiα (Top2α) activity, these results suggest a newly revealed, targetable aspect of ATC pathophysiology and a potential novel role for the involvement of Top2α in the resolution of R-loops. Citation Format: Ryan Mackay, Qinqin Xu, Paul Weinberger. Accumulation of DNA:RNA hybrids (R-loops) and cell death after YM155 treatment of anaplastic thyroid cancer hints at a novel therapeutic approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1838.

SAT-573 Characterization of the Gene Expression Program of a Mutant Thyroid Hormone Receptor in Anaplastic Thyroid Cancer

Thyroid cancer is the most commonly diagnosed endocrine malignancy. Although survival rates for the majority of thyroid cancer subtypes are high compared to other cancers, the de-differentiated anaplastic thyroid cancer (ATC) is particularly aggressive, with a median survival time of less than 6 months. Intriguingly, thyroid hormone receptor beta (TRβ) has been demonstrated to repress tumor growth in thyroid cancer models, but the mechanism is not well established. Additionally, phosphorylation of a tyrosine residue (Y406) is critical for the tumor suppressive phenotype of TRβ; mutation of this residue ablates TRβ-mediated tumor suppression (1). We hypothesized that the signaling mediated by wild type, but not Y406 TRβ defines the critical pathways for tumor suppression. We restored WT or Y406F TRβ expression by lentiviral transduction in an authenticated ATC cell line, SW1736. WT TRβ decreased cell proliferation, an effect amplified by T3. Y406F did not significantly reduce ATC cell proliferation and addition of T3 did not restore the antiproliferative effect implicating differential signaling. As we have previously identified TRβ suppression of the oncogene RUNX2 in thyroid cells (2), we determined the impact of Y406F TRβ on this signaling pathway. While Y406F TRβ reduced RUNX2 expression, the effect was less pronounced than noted with WT. Of importance, we previously demonstrated that the chromatin ATPase BRG1 cooperates with TRβ to reduce RUNX2 levels (3). Mutation of TRβ at Y406 eliminated this synergy. Finally, we determined the ligand-dependent and independent transcriptomes of WT and Y406F TRβ by RNA-sequencing. Critically, WT and Y406F TRβ both responded to T3 treatment, but exhibit different transcriptional profiles. Importantly, we were able to identify several tumor suppressor genes and putative tumor suppressor genes that were upregulated only in the WT-T3 treated cells including BRCA2, CHD1, and SEPT7. Utilizing Ingenuity Pathway Analysis, we determined repression of the epigenetic regulator KDM5B (a putative oncogene) and upregulation of CD24 activity (low in CD44+/CD24- cancer stem cells). These results define the TRβ transcriptome in aggressive thyroid cancer cells and reveal novel signaling in ATC. 1. Park JW, Zhao L, Webb P, Cheng S-y. Src-dependent phosphorylation at Y406 on the thyroid hormone receptor β confers the tumor suppressor activity. Oncotarget 2014;5:10002-16 2. Carr FE, Tai PW, Barnum MS, Gillis NE, Evans KG, Taber TH, et al. Thyroid Hormone Receptor-beta (TRbeta) Mediates Runt-Related Transcription Factor 2 (Runx2) Expression in Thyroid Cancer Cells: A Novel Signaling Pathway in Thyroid Cancer. Endocrinology 2016;157:3278-92 3. Gillis NE, Taber TH, Bolf EL, Beaudet CM, Tomczak JA, White JH, et al. Thyroid Hormone Receptor beta Suppression of RUNX2 is Mediated by Brahma Related Gene 1 Dependent Chromatin Remodeling. Endocrinology 2018

Overexpression of RYBP inhibits proliferation, invasion, and chemoresistance to cisplatin in anaplastic thyroid cancer cells via the EGFR pathway.

Ring1 and YY1 binding protein (RYBP), a new member of the polycomb group protein family, has been reported to play an important role in various biological processes. Recently, more and more studies have demonstrated an implication of RYBP in cancer development. However, the specific role of RYBP in anaplastic thyroid cancer (ATC) remains unknown. In this study, we investigated for the first time the expression pattern and biological functions of RYBP in ATC. We showed that RYBP was lowly expressed in ATC tissues and cell lines. We also found that overexpression of RYBP inhibited ATC cell proliferation, invasion, and cisplatin resistance. Furthermore, we observed that upregulation of RYBP decreased the phosphorylation of EGFR and ERK1/2 in ATC cells. Taken together, our data indicated that RYBP might be considered as a promising therapeutic target for the treatment of ATC.

Long noncoding RNA UCA1 promotes anaplastic thyroid cancer cell proliferation via miR‑135a‑mediated c‑myc activation.

The long non‑coding RNA (lncRNA), urothelial carcinoma‑associated 1 (UCA1), has been demonstrated to be dysregulated and serves a role in the progression of several cancer types. However, the exact effects and underlying molecular mechanisms of UCA1 in anaplastic thyroid cancer (ATC) remain unknown. The aim of the present study was to investigate the detailed function and the mechanism of UCA1 in the regulation of ATC cell progression. The present study identified that the expression levels of UCA1, in ATC cell lines and tissues, were significantly upregulated compared with normal human thyroid cell line and adjacent non‑cancerous tissues, respectively. UCA1 knockdown significantly inhibited ATC cell viability, proliferation, migration and invasion and the expression level of c‑myc proto‑oncogene (c‑myc) invitro, and suppressed ATC tumor growth invivo. In addition, using luciferase assays, it was confirmed that miR‑135a directly bound to UCA1 and the 3' untranslated region of c‑myc, and UCA1 competed with c‑myc for miR‑135a binding. miR‑135a inhibition may upregulate c‑myc expression, however, the upregulation of c‑myc may be partially reduced by short hairpin UCA1. The present results illustrated that UCA1 promoted ATC cell proliferation through acting as a competing endogenous RNA by binding miR‑135a. In conclusion, in the present study, UCA1 served as an oncogenic long non‑coding RNA promoting ATC cell proliferation and may be a potential target for human ATC treatment.

Vandetanib has antineoplastic activity in anaplastic thyroid cancer, in vitro and in vivo.

The antitumor activity of vandetanib [a multiple signal transduction inhibitor including the RET tyrosine kinase, epidermal growth factor receptor(EGFR), vascular endothelial growth factor(VEGF) receptor(VEGFR), ERK and with antiangiogenic activity], in primary anaplastic thyroid cancer(ATC) cells, in the human cell line 8305C [undifferentiated thyroid cancer(TC)] and in an ATC‑cell line (AF), was investigated in the present study. Vandetanib (1and100nM; 1, 10, 25and50µM) was tested by WST‑1, apoptosis, migration and invasion assays: in primary ATC cells, in the 8305C continuous cell line, and in AF cells; and in 8305C cells in CD nu/nu mice. Vandetanib significantly reduced ATC cell proliferation (P<0.01, ANOVA), induced apoptosis dose‑dependently (P<0.001, ANOVA), and inhibited migration (P<0.01) and invasion (P<0.001). Furthermore, vandetanib inhibited EGFR, AKT and ERK1/2 phosphorylation and downregulated cyclinD1 in ATC cells. In 8305C and AF cells, vandetanib significantly inhibited the proliferation, inducing also apoptosis. 8305C cells were injected subcutaneously in CD nu/nu mice and tumor masses became detectable after 30days. Vandetanib (25mg/kg/day) significantly inhibited tumor growth and VEGF‑A expression and microvessel density in 8305C tumor tissues. In conclusion, the antitumor and antiangiogenic activity of vandetanib is very auspicious in ATC, opening the way to a future clinical evaluation.

Lenvatinib exhibits antineoplastic activity in anaplastic thyroid cancer in vitro and in vivo

Lenvatinib is an oral, multitargeted tyrosine kinase inhibitor(TKI) of VEGFR1-VEGFR3, FGFR1-FGFR4, PDGFRα, RET and v-kit Hardy-Zuckerman4 feline sarcoma viral oncogene homolog(KIT) signaling networks involved in tumor angiogenesis. We have evaluated the antitumor activity of lenvatinib in primary anaplastic thyroid cancer (ATC) cells, in the human cell line 8305C (undifferentiated thyroid cancer) and in an ATC-cell line (AF). The AF cell line was obtained from the primary ATC cultures and was the one that grew over 50passages. The effect of lenvatinib (1 and 100nM; and 1, 10, 25 and 50µM) was investigated in primary ATC, 8305C and AF cells as well as in AF cells in CDnu/nu mice. Lenvatinib significantly reduced ATC cell proliferation(P<0.01, ANOVA) and increased the percentage of apoptotic ATC cells (P<0.001, ANOVA). Furthermore, lenvatinib inhibited migration (P<0.01) and invasion(P<0.001) in ATC. In addition, lenvatinib inhibited EGFR, AKT and ERK1/2 phosphorylation and downregulated cyclinD1 in the ATC cells. Lenvatinib also significantly inhibited 8305C and AF cell proliferation, increasing apoptosis. AF cells were subcutaneously injected into CDnu/nu mice and tumor masses were observed 20days later. Tumor growth was significantly inhibited by lenvatinib(25mg/kg/day), as well as the expression of VEGF-A and microvessel density in the AF tumor tissues. In conclusion, the antitumor and antiangiogenic activities of lenvatinib may be promising for the treatment of anaplastic thyroid cancer, and may consist a basis for future clinical therapeutic applications.

Effects of HuR downregulation on anaplastic thyroid cancer cells.

Anaplastic thyroid cancer (ATC) constitutes one of the most aggressive types of human solid cancer, and is characterized by the absence of thyroid differentiation features and a marked degree of invasiveness. We have previously demonstrated that the RNA-binding protein Hu antigen R (HuR) is overexpressed in thyroid carcinoma; thus, the biological role of this RNA-binding protein was investigated in the present study using the ATC cell lines SW1736 and 8505C. In both cell lines, HuR protein levels were higher compared with in the non-tumorigenic thyroid cell line Nthy-ori-3.1. HuR silencing by RNA interference in both ATC cell lines decreased cell viability, increased apoptosis rates and reduced the capability to form colonies in soft agar. Thus, HuR plays an important role in the proliferation and aggressiveness of ATC cells. The histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) was able to reduce the viability of ATC cells. The results demonstrated that SAHA was able to decrease HuR expression in SW1736 and 8505C cells. Furthermore, since it is known that the transcription factor nuclear factor (NF)-κB modulates HuR expression, whether SAHA affects the nuclear (active) fraction of NF-κB in ATC cells was investigated. The data suggested that SAHA decreases ATC cell viability by reducing the active form of NF-κB, which, in turn, modulates HuR expression.