Anaplastic Thyroid Carcinoma Cell Lines Research Articles

MiR-381-3p attenuates doxorubicin resistance in human anaplastic thyroid carcinoma via targeting homeobox A9.

Abnormal microRNA (miR) expression has frequently been reported to be implicated in cancer-related drug resistance. Herein, we planned to investigate whether miR-381-3p contributes to doxorubicin (DOX) resistance in anaplastic thyroid carcinoma (ATC). DOX-resistant ATC tissues and cell lines were prepared to detect miR-381-3p and homeobox A9 (HOXA9) expression. CCK8, transwell and TUNEL assays were performed to evaluate cell proliferation, migration and invasion, and apoptosis in in vitro experiments. HOXA9 expression is intensively expressed in ATC tissues compared with benign thyroid tissues. Compared with parental ATC cell lines, HOXA9 protein expression is significantly up-regulated in DOX-resistant SW1736 and CAL62 cells. The knockdown of HOXA9 leads to growth inhibition and apoptosis of DOX-resistant SW1736 and CAL62 cells. Our results also indicate a significant decrease in miR-381-3p expression levels in DOX-resistant ATC tissues and cell lines. miR-381-3p may function as a tumour suppressor to impede proliferation, migration and invasion and induce apoptosis of DOX-resistant SW1736 and CAL62 cells by inhibiting HOXA9 protein expression. Our results present a novel signalling axis miR-381-3p/HOXA9 that mediates DOX resistance in ATC. miR-381-3p and HOXA9 may be promising molecular targets for preventing ATC progression and drug resistance.

Exosome-mediated delivery of SCD-1 siRNA promoted the death of anaplastic thyroid carcinoma cells via regulating ROS level.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive cancers in the world. Stearoyl-CoA desaturase-1 (SCD-1) is one of major enzymes in the de novo synthesis of fatty acids and is related to cancer aggressiveness and poor patient prognosis. The study aimed to construct exosomes loaded SCD-1 interference, investigate its effects and mechanisms on the cell proliferation and apoptosis of ATC cells. The expressions of SCD-1 in normal thyroid cell line and ATC cell lines were determined by qRT-PCR and western blotting, respectively. Exosomes were prepared and purification then loaded with SCD-1 siRNA by electroporation and observed by transmission electron microscopy. Higher SCD-1 mRNA and protein levels were found in ATC cell lines compared than normal thyroid cell line (P < 0.05), and both Hth-7 and FRO cells could uptake PKH67-labeled exosomes. The effects of exosomes loaded SCD-1 siRNA on ATC cells were measured by CCK8 assay and apoptosis detection kit. When compared with control group, the cell viability significantly decreased in both two ATC cell lines taken up exosomes loaded SCD-1 siRNA (P < 0.001), and apoptotic and necrotic cells obviously increased (P < 0.05). In order to explore the mechanism of exosomes loaded SCD-1 on ATC, the ROS level was detected by fluorescence reagent. It was found that exosomes loaded SCD-1 siRNA significantly increased intracellular ROS level of ATC cells (P < 0.05). Exosomes loaded SCD-1 siRNA inhibited ATC cellular proliferation and promoted cellular apoptosis, and the mechanisms involved maybe the regulation of fatty acids metabolism and ROS level. Our study provides a promising therapeutic strategy for ATC.

Cytotoxicity, Apoptosis induction and change of p53, PARP, p21 and Bcl-2 genes expression in the human anaplastic thyroid carcinoma cells line (SW-1736) with curcumin

Anaplastic thyroid carcinoma is highly invasive with a poor response to a treatment. In this study, curcumin bioavailability and its effects on apoptosis induction and selected genes expression of the human anaplastic thyroid carcinoma cell line (SW-1736) were examined. SW-1736 cells were incubated for 24 and 48 hours with different concentrations of curcumin 2.5, 5, 7.5 and 10 μM to examine bioavailability, and for 24, 48 and 72 hours with concentrations of 2.5, 5, 7.5, 10 μM and 2.5, 5 and 10 μM respectively to examine apoptosis and the expression of p53, PARP, p21 and Bcl-2 genes. Then, bioavailability was analyzed by MTS kit, apoptosis was analyzed by flow- cytometry using Annexin V-FITC/PI kit and the expression of p53, PARP, p21 and Bcl-2 genes were analyzed by Real Time PCR. ANOVA test and SPSS 16 software were used for statistical analysis. The results indicate that curcumin at the concentration of 7.5 μM has significantly decreased bioavailability in anaplastic thyroid cells in comparison with other treatments at both incubation periods. Induction of apoptosis with increasing concentration of curcumin in dose and time dependent manner increased in this cell line. Also, treatment with curcumin significantly decreased the expression of Bcl-2 gene and increased the expression of p53, PARP and p21 genes in some experimental groups compared to the control group. Curcumin inhibited the growth, proliferation and invasion of anaplastic thyroid cancer cells through altering the expression of the genes involved in the apoptosis process.

Identification of an immune-related signature indicating the dedifferentiation of thyroid cells

BackgroundImmune cells account for a large proportion of the tumour microenvironment in anaplastic thyroid carcinomas (ATCs). However, the expression pattern of immune-related genes (IRGs) in ATCs is unclear. Our study aimed to identify an immune-related signature indicating the dedifferentiation of thyroid cells.MethodsWe compared the differences in thyroid differentiation score (TDS), infiltration of immune cells and enriched pathways between ATCs and papillary thyroid carcinomas (PTCs) or normal thyroid tissues in the Gene Expression Omnibus database. Univariate and multivariable Cox analyses were used to screen prognosis-associated IRGs in The Cancer Genome Atlas database. After constructing a risk score, we investigated its predictive value for differentiation and survival by applying receiver operating characteristic and Kaplan–Meier curves. We further explored its associations with important immune checkpoint molecules, infiltrating immune cells and response to immunotherapy.ResultsCompared with PTCs or normal thyroid tissues, ATCs exhibited lower TDS values and higher enrichment of immune cells and activation of the inflammatory response. The quantitative analyses and immunohistochemical staining validated that most ATC cell lines and ATC tissues had higher expression of MMP9 and lower expression of SDC2 than normal thyroid samples and PTC. Higher risk scores indicates dedifferentiation and a worse prognosis. Additionally, the risk score was positively correlated with the immune checkpoint molecules PDL1, CTLA4, IDO1, and HAVCR2 and infiltration of multiple immune cells. Importantly, we found that the samples with higher risk scores tended to have a better response to immunotherapy than those with lower scores.ConclusionOur findings indicate that the risk score may not only contribute to the determination of differentiation and prognosis of thyroid carcinomas but also help the prediction of immune cells infiltration and immunotherapy response.

Effects of melatonin on the toxicity and proliferation of human anaplastic thyroid cancer cell line

BackgroundThyroid carcinoma is the most common endocrine malignancy and anaplastic thyroid carcinoma (ATC) is a rare but most aggressive cancer. Melatonin has enhanced or induced apoptosis in many different cancer cells, however, there has not been any study on the effects of melatonin in the treatment of ATC. In this study, we examined the effect of melatonin on cytotoxicity in the human ATC cell line. Materials and methodsCultured ATC cells were treated at melatonin concentrations 0.6, 1, 4, 16, 28 mM for 24 h. The MTT assay was performed to examine cell viability. Cytotoxicity was assayed with the determination of lactic dehydrogenase (LDH) activity. Apoptosis was detected by acridine orange/ethidium bromide and Hoechst 33342 staining. Giemsa staining is considered for evaluating the morphological changes of ATC cells. The reproductive ability of cells to form a colony was evaluated by the clonogenic assay. ResultsResults showed that melatonin could significantly decrease cell viability and the lowest cell viability was observed at 28 mM, 10.26 % ± 0.858 versus control. Similar results were obtained when analyzing LDH activity. The highest LDH levels were observed at 16 and 28 mM (546.08 ± 4.66, 577.82 ± 3.14 munit/mL versus control) that confirmed the occurrence of late apoptosis. The clonogenic assay showed that cells at the high concentration of melatonin (16 and 28 mM) don't enable to form the colony that approved the occurrence of reproductive death. ConclusionOur results showed a dose-dependent cytotoxic effect of melatonin on ATC cells that significantly decreased cell viability and induced cell reproductive death at the concentration greater than 1 mM and findings suggested that MLT might be useful as an adjuvant in ATC therapy.

Comparison of Proteomics Profiles Between Xenografts Derived from Cell Lines and Primary Tumors of Thyroid Carcinoma.

Patient-consistent xenograft model is a challenge for all cancers but particularly for thyroid cancer, which shows some of the greatest genetic divergence between human tumors and cell lines. In this study, proteomic profiles of tumor tissues from patients, included anaplastic thyroid carcinoma (ATC) and papillary thyroid carcinoma, and xenografts (8305C, 8505C, FRO, BAPAP and IHH4) were obtained using HPLC-tandem mass spectrometry and compared based on all proteins detected (3,961), cancer-related proteins and druggable proteins using pairwise Pearson's correlation analysis. The human tissue showed low proteomic similarity to the ATC cell lines (8305C, r = 0.344-0.416; 8505C, 0.47-0.579; FRO, 0.267-0.307) and to PTC cell lines (BCPAP, 0.303-0.468; IHH4, 0.262-0.509). Human tissue showed the following similarity to cell lines at the level of 135 cancer-related pathways. The ATC cell lines contained 47.4% of the cancer-related pathways (19.26%-33.33%), while the PTC cell lines contained 40% (BCPAP, 25.93%; IHH4, 28.89%). In patient tumor tissues, 44-60 of 76 and 52-53 of 93 druggable proteins were identified in ATC and PTC tumors, respectively. Ten and 29 druggable proteins were not identified in any of the ATC and PTC xenografts, respectively. We provide a reference for CDX selecting in in vivo studies of thyroid cancer.

Synergistic effect of 17-allylamino-17-demethoxygeldanamycin with dehydroxymethylepoxyquinomicin on the human anaplastic thyroid carcinoma cell line KTC2

The use of targeted inhibitors has shown promise as an effective approach in cancer therapy. However, targeted therapies based only on one drug, such as 17-allylamino-17-demethoxygeldanamycin (17-AAG), have limited success, partly because cancer cells engage alternate pathways for survival and proliferation. In the present study, we evaluated whether dehydroxymethylepoxyquinomicin (DHMEQ), a nuclear factor ?B (NF-?B) inhibitor, can enhance the antitumor activities of 17-AAG, a 90 kDa heat shock protein (Hsp90) inhibitor, in the anaplastic thyroid cancer cell line KTC2. We examined the effect of combined drug treatment vs single drug treatment on cell survival. Isobologram analysis was performed to distinguish the additive vs synergistic effects of the drug combination. Western blotting was performed to investigate apoptosis markers: caspase 3, poly(ADP-ribose) polymerase-one (PARP-1), B-cell lymphoma-extra large (Bcl-XL), X-linked inhibitor of apoptosis (XIAP) and cellular inhibitor of apoptosis 2 (cIAP-2). Compared to monotherapy, the combined treatment enhanced growth-inhibitory effects in a synergistic manner and strongly potentiated apoptosis. These results demonstrate the first in vitro evidence that a combination of Hsp90 and NF-?B inhibitors is a more effective modality for inhibiting cell proliferation and survival in anaplastic thyroid carcinoma cells than either agent alone, warranting further investigations.

Regulatory Single Nucleotide Polymorphism Increases TERT Promoter Activity in Thyroid Carcinoma Cells

Background/Aim: The telomerase reverse transcriptase (TERT) promoter has a regulatory single nucleotide polymorphism (rSNP), rs2853669, and occasionally shows point mutations C228T and C250T. Although C228T and C250T have been well examined to increase TERT promoter activity and are known as risk factors for thyroid carcinoma, the significance of rs2853669 has not been well investigated. This study aimed to clarify the influence of rs2853669 on TERT promoter activity in thyroid carcinoma cells. Materials: Seven of 8 examined thyroid cell lines had rs2853669, 5 had C228T, and 1 had C250T. Results: Three papillary thyroid carcinoma cell lines, harboring both rs2853669 and C228T, showed higher TERT mRNA expression on real-time PCR than the other cell lines. Anaplastic thyroid carcinoma cell lines, in contrast, showed variable TERT mRNA expression depending on the combination of rs2853669, C228T, and C250T. Luciferase assays, performed to compare the influences of rs2853669, C228T, and C250T on TERT promoter activity in thyroid carcinoma, showed that rs2853669, as well as C228T, increased the promoter activity, and the combination of rs2853669 and C228T increased the promoter activity even more strongly than C228T alone. Conclusion: We conclude that the presence of rs2853669 within the TERT promoter could be as significant as the C228T mutation in thyroid carcinoma.

EIF5A2 Is Highly Expressed in Anaplastic Thyroid Carcinoma and Is Associated With Tumor Growth by Modulating TGF- Signals.

Anaplastic thyroid carcinoma (ATC) is resistant to standard therapies and has no effective treatment. Eukaryotic translation initiation factor 5A2 (EIF5A2) has shown to be upregulated in many malignant tumors and proposed to be a critical gene involved in tumor metastasis. In this study, we aimed to investigate the expression status of EIF5A2 in human ATC tissues and to study the role and mechanisms of EIF5A2 in ATC tumorigenesis in vitro and in vivo. Expression of EIF5A2 protein was analyzed in paraffin-embedded human ATC tissues and adjacent nontumorous tissues (ANCT) (n = 24) by immunochemistry. Expressions of EIF5A2 mRNA and protein were analyzed in fresh-matched ATC and ANCT (n = 23) and ATC cell lines by real-time polymerase chain reaction (PCR) and Western blotting. The effect of targeting EIF5A2 with short hairpin RNA (shRNA) or EIF5A2 overexpression on the ATC tumorigenesis and TGF-β/Smad2/3 signals in vitro and in vivo was investigated. Expression of EIF5A2 was significantly upregulated in ATC tissues and cell lines compared with ANCT and normal follicular epithelial cell line. Functional studies found that targeting EIF5A2 induced SW1736 cell death in vitro and in vivo, followed by significantly downregulated phosphorylation of Smad2/3 (p-Smad2/3) in SW1736 cells at the protein level. Ectopic expression of EIF5A2 could promote 8505C cell growth in vitro and in vivo, followed by significantly upregulated p-Smad3 at the protein level. Recombinant human TGF-β1 (hTGF-β1) treatment decreased the antiproliferative activity of the EIF5A2 downexpressing 8505C cells through reversing pSmad2/3. Using the specific inhibitor SB431542 to block TGF-β pathway or Smad3 siRNA to knock down Smad3 increased the antiproliferative activity of the EIF5A2-overexpressing 8505C cells through inhibiting pSmad2/3. Our findings indicated that EIF5A2 controled cell growth in ATC cells, and EIF5A/TGF-β/Smad2/3 signal may be a potential therapeutic target for ATC treatment.

3D polymeric supports promote the growth and progression of anaplastic thyroid carcinoma

Anaplastic thyroid carcinoma (ATC) is a rare and aggressive malignancy that accounts for the majority of deaths from all thyroid cancers. ATC exhibits invasiveness and highly resistance to conventional therapies which include cytotoxic chemotherapy, the combination of BRAF and MEK inhibition and, more recently, immunotherapies, that have shown promising but still limited results. A growing knowledge on ATC tumor biology is needed for developing more effective therapies with significant better survival. Researchers have begun to utilize 3D models to culture cancer cells for in vitro studies. In this work, C643 ATC cell line was cultured on polymeric scaffolds with high-interconnected porous matrix. They exhibited distinct viability, proliferation and 3D morphology similar to an in vivo solid tumor mass. We also carried out quantitative real-time PCR experiments for monitoring Cancer Stem Cells enrichment, since they are most probably the cause of tumor resistance, reoccurrence and metastasis. The same tests were performed after cell treatment with the chemotherapic Doxorubicin. An up-regulation of the analyzed stem-cell markers confirmed the high resistance to treatment of these cell line with respect to conventional drugs. In conclusion, 3D scaffolds could be an ideal platform for studying the mechanisms that regulate ACT growth and survival and also improving novel therapeutic approaches for treatment-resistant thyroid cancer.

Non‑invasive monitoring of paclitaxel and lenvatinib efficacy against anaplastic thyroid cancer in orthotopic SCID mouse models using small‑animal FDG‑PET/CT.

Anaplastic thyroid carcinoma (ATC) is a rare type of thyroid carcinoma with a poor prognosis. Thus, suitable preclinical tumor models are required for the development of new ATC therapies. In the present study, orthotopic tumor xenograft models were established using ATC cell lines and SCID mice, and tumor invasion and the effects of anticancer drugs were evaluated using positron emission tomography/computed tomography (PET/CT) to repeatedly and non‑invasively monitor these models. Three ATC cell lines (8305c, 8505c, and ACT‑1) were used. Their sensitivities to two anticancer drugs (paclitaxel and lenvatinib) were investigated. The 8505c cell line was orthotopically implanted into SCID mice, which were then divided into three groups: No chemotherapy, paclitaxel (5mg/kg, administered intraperitoneally, every week), and lenvatinib (5mg/kg, oral route, every day) groups. PET/CT was performed and tumor growth and the effects of anticancer drugs based on tumor volume and fludeoxyglucose (FDG) uptake were evaluated. 8505c cells exhibited the highest sensitivity to the anticancer drugs. In mice implanted with 8505c cells, continuous increases in FDG uptake associated with tumor growth were detected on PET/CT in the group that received no chemotherapy. The tumor volume and FDG uptake increased by 91.5‑ and 2.4‑fold, respectively, within 2weeks. The increase observed in tumor volume was 26.9‑ and 12.2‑fold in the paclitaxel and lenvatinib groups, respectively, within 2weeks. Furthermore, the increase in FDG uptake was 1.8 and 1.6‑fold in the paclitaxel and lenvatinib groups, respectively, within 2weeks. In our orthotopic SCID mouse model, tumor growth and the effects of anticancer drugs were repeatedly and non‑invasively monitored using PET/CT. The present method is useful for the development of new ATC treatments.

Down-Regulation of the Mammalian Target of Rapamycin (mTOR) Pathway Mediates the Effects of the Paeonol-Platinum(II) Complex in Human Thyroid Carcinoma Cells and Mouse SW1736 Tumor Xenografts.

BackgroundThis study aimed to investigate the effects of the paeonol-platinum(II) (PL-Pt[II]) complex on SW1736 human anaplastic thyroid carcinoma cell line and the BHP7-13 human thyroid papillary carcinoma cell line in vitro and on mouse SW1736 tumor xenografts in vivo.Material/MethodsThe cytotoxic effects of the PL-Pt(II) complex on SW1736 cells and BHP7-13 cells was measured using the MTT assay. Western blot measured the expression levels of cyclins, cell apoptotic proteins, and signaling proteins. DNA content and apoptosis were detected by flow cytometry. SW1736 cell thyroid tumor xenografts were established in mice followed by treatment with the PL-Pt(II) complex.ResultsTreatment of the SW1736 and BHP7-13 cells with the PL-Pt(II) complex reduced cell proliferation in a dose-dependent manner, with an IC50 of 1.25 μM and 1.0 μM, respectively, and increased the cell fraction in G0/G1phase, inhibited p53, cyclin D1, promoted p27 and p21 expression, and significantly increased the sub-G1 fraction. Treatment with the PL-Pt(II) complex increased caspase-3 degradation, reduced the expression of p-4EBP1, p-4E-BP1 and p-S6, and reduced the expression of p-ERK1/2 and p-AKT. Treatment with the PL-Pt(II) complex reduced the volume of the SW1736 mouse tumor xenografts on day 14 and day 21, and reduced AKT phosphorylation and S6 protein expression and increased degradation of caspase-3.ConclusionsThe cytotoxic effects of the PL-Pt(II) complex in human thyroid carcinoma cells, including activation of apoptosis and an increased sub-G1 cell fraction of the cell cycle, were mediated by down-regulation of the mTOR pathway.

Identification of key candidate genes and pathways in anaplastic thyroid cancer by bioinformatics analysis.

Anaplastic thyroid carcinoma (ATC) is a refractory and poor prognosis tumor Present study aimed to investigate the underlying biological functions and pathways involved in the development of ATC and to identify potential hub genes and candidate biomarkers of ATC. Bioinformatics analyses were performed to identify the differentially expressed genes (DEGs) between ATC tissue samples and adjacent normal tissue samples. Protein-protein interaction (PPI) networks of the DEGs were constructed using Search Tool for the Retrieval of Interacting Genes online tool and Cytoscape software and divided into sub-networks using the Molecular Complex Detection (MCODE) plug-in. DEGs in each module was analyzed by enrichment analysis of the KEGG Orthology Based Annotation System (KOBAS) web software version 3.0. Eventually, the hub genes from bioinformatics analysis were verified by qRT-PCR assay in different ATC cell lines. Thirty hub genes were selected and three modules were built by the Cytoscape software from the PPI network. Seven genes (CDK1, CCNB2, BUB1B, CDC20, RRM2, CHEK1 and CDC45) were screened from thirty hub genes. Enrichment analysis showed that these hub genes were primarily accumulated in 'cell cycle', 'p53 signaling pathway', 'viral carcinogenesis', 'pyrimidine metabolism' and 'ubiquitin mediated proteolysis'. The results of qRT-PCR indicated that seven hub genes were unregulated in three ATC cell lines compared with normal thyroid gland cell. These findings suggest that CDK1, CCNB2, BUB1B, CDC20, RRM2, CHEK1 and CDC45 may serve as novel diagnosis biomarkers and potential therapeutic target for ATC.

MiR-19a Is Involved In Progression And Malignancy Of Anaplastic Thyroid Cancer Cells.

BackgroundMicroRNAs (miRNAs) are endogenous, single-stranded, non-coding RNAs acting as negative regulators of gene expression involved in a number of physiological processes. MiRNAs’ expression is commonly dysregulated in many types of human tumor diseases and cancers, including thyroid cancers, and is often involved in tumor initiation and progression. miR-19a, a member of miR-17-92 cluster, has been demonstrated to promote cell growth in anaplastic thyroid cancer (ATC), the most advanced and aggressive thyroid cancer.PurposeIn this work, we investigate the potential contribution of miR-19a in thyroid cancer cells poor prognosis and de-differentiation.MethodsWe directly modulated the expression of miR-19a in papillary (PTC) and anaplastic thyroid carcinoma cell lines through transfection of specific miR-19a mimic or inhibitor. Further, we performed gene expression analysis of specific genes to evaluate miR-19a association with cell cycle, differentiation, and poor prognosis.ResultsOur data indicate that miR-19a overexpression in PTC cells significantly promotes cell growth, decreases the expression of differentiation genes and activates poor prognosis genes. Its inhibition in ATC cells reduces cell proliferation and the expression of genes related to poor prognosis but does not affect differentiation.ConclusionOur findings reveal the existence of functional associations between miR-19a expression and thyroid cancer progression and malignancy suggesting miR-19a as a novel candidate therapeutic target for ATC.

LncRNA NEAT1 enhances the resistance of anaplastic thyroid carcinoma cells to cisplatin by sponging miR‑9‑5p and regulating SPAG9 expression.

Anaplastic thyroid carcinoma (ATC) has a poor prognosis due to its resistance to all conventional treatments. The long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) serves a critical role in cancer chemoresistance; however, whether NEAT1 is associated with chemoresistance of ATC remains unclear. In the present study, reverse transcription-quantitative PCR assays were performed to detect the expression levels of NEAT1, microRNA (miR)-9-5p and sperm-associated antigen 9 (SPAG9). Western blot analysis was conducted to assess the protein expression levels of p62, microtubule-associated proteins 1A/1B light chain 3B and SPAG9. Cell proliferation was detected using the Cell Counting kit-8 assay, and cell apoptosis was determined by flow cytometry. Dual-luciferase reporter and RNA immunoprecipitation assays were performed to verify the interaction between NEAT1 and miR-9-5p, or miR-9-5p and SPAG9. Furthermore, an animal model was used to investigate the regulatory effects of NEAT1 on cisplatin (DDP)-resistance in tumors in vivo. The present results demonstrated that NEAT1 was upregulated in ATC tissues and cell lines, and NEAT1 silencing resulted in decreased DDP-resistance of ATC cells. In addition, NEAT1 suppressed miR-9-5p expression by binding to miR-9-5p and SPAG9 was a direct target of miR-9-5p. miR-9-5p overexpression sensitized ATC cells to DDP. Notably, NEAT1 silencing exerted its inhibitory effect on DDP-resistance of ATC via the miR-9-5p/SPAG9 axis in vitro and in vivo. In conclusion, the present study demonstrated that NEAT1 silencing ameliorated DDP-resistance of ATC, at least in part by reducing miR-9-5p sponging and regulating SPAG5 expression; therefore, NEAT1 may be considered a potential therapeutic target of ATC.

Abstract 1164: Expression of Notch and Nodal embryonic components in BRAF- and RAS-like thyroid cancer cell lines

Abstract TCGA genomic study in papillary thyroid carcinoma (PTC) samples highlights two groups of tumors: BRAF-like and RAS-like, whose genotype might influence its biological behavior. The mechanisms behind progressive loss of thyroid differentiation, observed mainly in the most aggressive histotype anaplastic thyroid carcinoma (ATC), could be associated with the reactivation of embryonic signaling and acquisition of stem cell-like phenotype. In this study we focused on Notch and Nodal embryonic signaling pathways, which are crucial for stemness maintenance in embryonic cells and has also been linked to cancer cell stemness. By assessing the expression of Notch and Nodal pathway components in BRAF- and RAS-like cell lines, we sought to investigate if the reactivation of these pathways is involved in dedifferentiation mechanisms observed in ATC. For that purpose, RNA and protein were extracted from three PTC (TPC-1, BCPAP and K1), six ATC (KTC2, SW1736, C643, Hth7, Hth74 and Hth83) and non-tumoral (Nthy-ori 3-1) cell lines. For gene expression analysis, cDNA generate from mRNA was amplified by qPCR using specific primers for NOTCH1, NOTCH4, HES1, NUMB, CER1 and LEFTY1/2. For protein expression analysis, SDS-PAGE-fractionated proteins were transferred onto nitrocellulose membrane, incubated with anti-Nodal antibody and visualized by chemoluminescence. NOTCH1 was downregulated in BRAF-like PTC cell lines, while slightly overexpressed in RET/PTC-containing TPC-1. On the other hand, NOTCH1 was overexpressed in Hth83 and C643 ATC lines, both HRAS-mutated, and also in BRAF-mutated SW1736. NOTCH4 was overexpressed in PI3K-mutated K1 and all ATC cell lines. Transcription repressor HES1, which prevents cell cycle arrest by inhibiting p27, was highly expressed in all cell lines compared to Nthy. Levels of NUMB expression were elevated in all ATC cell lines with no difference between BRAF- and RAS-mutated, but were inversely related to those of HES1, as expected for a Notch pathway inhibitor. CER1 and LEFTY1/2, which encode Nodal inhibitors, were highly expressed in ATC cell lines (4/6 and 5/6, respectively) but with no apparent distinction between BRAF- and RAS-mutated cells. At the same time, Nodal protein levels are only upregulated in TP53-mutated BCPAP and C643, suggesting that alterations in MAPK components are not sufficient to promote Nodal overexpression. Our results show re-expression of Notch embryonic signaling components in both PTC and ATC cell lines with either BRAF or RAS oncogenes. Moreover, additional genetic alterations such as in PI3K and TP53 might also interfere with the expression of both Notch and Nodal components. We therefore highlight the importance of tumor genotype, which might determine the modulation of embryonic-related genes and therefore contribute to the undifferentiated phenotype in thyroid cancer. Financial support: FAPESP and CNPq. Citation Format: Paola M. Dantonio, Cesar Seigi Fuziwara, Kelly C. Saito, Edna T. Kimura. Expression of Notch and Nodal embryonic components in BRAF- and RAS-like thyroid cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1164.

Targeting CD47 in Anaplastic Thyroid Carcinoma Enhances Tumor Phagocytosis by Macrophages and Is a Promising Therapeutic Strategy.

Background: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human cancers, with a median survival of only three to six months. Standard treatment options and even targeted therapies have so far failed to improve long-term overall survival. Thus, novel treatment modalities for ATC, such as immunotherapy, are urgently needed. CD47 is a “don't eat me” signal, which prevents cancer cells from phagocytosis by binding to signal regulatory protein alpha on macrophages. So far, the role of macrophages and the CD47–signal regulatory protein alpha signaling axis in ATC is not well understood.Methods: This study analyzed 19 primary human ATCs for macrophage markers, CD47 expression, and immune checkpoints by immunohistochemistry. ATC cell lines and a fresh ATC sample were assessed by flow cytometry for CD47 expression and macrophage infiltration, respectively. CD47 was blocked in phagocytosis assays of co-cultured macrophages and ATC cell lines. Anti-CD47 antibody treatment was administered to ATC cell line xenotransplanted immunocompromised mice, as well as to tamoxifen-induced ATC double-transgenic mice.Results: Human ATC samples were heavily infiltrated by CD68- and CD163-expressing tumor-associated macrophages (TAMs), and expressed CD47 and calreticulin, the dominant pro-phagocytic molecule. In addition, ATC tissues expressed the immune checkpoint molecules programmed cell death 1 and programmed death ligand 1. Blocking CD47 promoted the phagocytosis of ATC cell lines by macrophages in vitro. Anti-CD47 antibody treatment of ATC xenotransplanted mice increased the frequency of TAMs, enhanced the expression of macrophage activation markers, augmented tumor cell phagocytosis, and suppressed tumor growth. In double-transgenic ATC mice, CD47 was expressed on tumor cells, and blocking CD47 increased TAM frequencies.Conclusions: Targeting CD47 or CD47 in combination with programmed cell death 1 may potentially improve the outcomes of ATC patients and may represent a valuable addition to the current standard of care.

Transcript-level regulation of MALAT1-mediated cell cycle and apoptosis genes using dual MEK/Aurora kinase inhibitor "BI-847325" on anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is the most lethal malignancy in thyroid carcinomas. Long non-coding RNAs (lncRNAs) are a member of non-coding RNAs, regulating the expression of gene. Metastasis-associated lung adenocarcinoma transcript1 (MALAT1) is an onco-lncRNA that is overexpressed in several carcinomas including ATC. Evidence showed that MALAT1 has a crucial function in apoptosis, and cell cycle progression. In order to take advantage of 3D cell culture system in cancer investigation, we have used a 3D in vitro ATC model to determine the effect of dual MEK/Aurora kinase inhibitor BI-847325 anticancer drug on the fundamental molecular mechanisms of MALAT1-mediated gene regulation in ATC. In this study, ATC cell lines (C643 and SW1736) were grown in alginate scaffold. Encapsulated cells were treated by BI-847325. Changes in expression of MALAT1, Mcl1, miR-363-3p, and cyclinD1 were measured by qRT-PCR. MALAT1 gene expression following BI-847325 treatment was significantly downregulated in C643 and SW1736 cell lines. Reversely, miR-363-3p expression was significantly upregulated by BI-847325 in both ATC cell lines. Mcl1 expression was significantly downregulated after treatment in C643 cell lines. Moreover, the expression of this gene was not significantly reduced following BI-847325 treatment in SW1736 cell line. Additionally, cyclin D1 expression was significantly downregulated after treatment in both ATC cell lines. Altogether, the result of this study was the first report of MALAT1's molecular function in ATC and suggested that BI-847325 which inhibits both MEK and Aurora kinase family could be effective against ATC by regulating the genes involved in cell cycle and apoptosis including MALAT1and its downstream genes. Graphical abstract Schematic representation of the biological role of MALAT1 in cyclin D1, miR-363-3p and Mcl1 gene regulations. Stimulation of receptor tyrosine kinase (RTK) by growth factors (GFs) phosphorylates RAS that subsequently activates RAF. Then, RAF phosphorylates MEK. Consequently, activated MEK phosphorylates ERK downstream effector, leading to the MALAT1 gene expression. MALAT1 is a negative regulator of Mcl1 mRNA by sponging of miR-363-3p. In addition, MALAT1 leads to Axin1 and APC downregulation and Wnt/β-catenin signaling pathway activation. Stable β-catenin translocates from the cytoplasm to the nucleus and promotes cyclin D1 gene expression.

Targeting the BRAF Signaling Pathway in CD133pos Cancer Stem Cells of Anaplastic Thyroid Carcinoma

Background:Cancer stem cells (CSCs) with a self-renewal ability in tumor cells population, execute a pivotal function in tumorigenesis, retrogression, and metastasis of malignant cancers such as anaplastic thyroid carcinoma (ATC).Materials and Methods:In this study, we isolated CSCs subpopulation with CD133 surface marker from three ATC cell lines by magnetic cell sorting assay. After confirming the segregation by the flow cytometry method, BRAF and sodium-iodide symporter (NIS) genes were investigated in them before and after incubation with BRAF inhibitor. Also, we evaluated the NIS protein expression and localization.Results:Established upon q-RT PCR data, when compared to human normal thyrocytes, the BRAFV600E gene was over-expressed in CD133pos cells (>1705.99 ± 55.55 fold, Mean ± SEM, n=3, P- value<0.05), whilst the expression of NIS gene was very restricted (< 0.0008 ± 5.43 fold, Mean ± SEM, n=3, P- value<0.05) in them. Also, our results showed that BRAF inhibition affected NIS protein expression and localization.Conclusions:Current study showed that the differentiate genes/proteins expression can be induced in the CSCs via focus on signal transduction pathways and targeting their molecules, that are involved in expression of these genes/proteins. Therefore, attention to targeting CSCs along with routine thyroid cancer therapy, can help to ATC treatment.

Integrated Bioinformatics Analysis of Master Regulators in Anaplastic Thyroid Carcinoma

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and rapidly lethal tumors. However, limited advances have been made to prolong the survival and to reduce the mortality over the last decades. Therefore, identifying the master regulators underlying ATC progression is desperately needed. In our present study, three datasets including GSE33630, GSE29265, and GSE65144 were retrieved from Gene Expression Omnibus with a total of 32 ATC samples and 78 normal thyroid tissues. A total of 1804 consistently changed differentially expressed genes (DEGs) were identified from three datasets. KEGG pathways enrichment suggested that upregulated DEGs were mainly enriched in ECM-receptor interaction, cell cycle, PI3K-Akt signaling pathway, focal adhesion, and p53 signaling pathway. Furthermore, key gene modules in PPI network were identified by Cytoscape plugin MCODE and they were mainly associated with DNA replication, cell cycle process, collagen fibril organization, and regulation of leukocyte migration. Additionally, TOP2A, CDK1, CCNB1, VEGFA, BIRC5, MAPK1, CCNA2, MAD2L1, CDC20, and BUB1 were identified as hub genes of the PPI network. Interestingly, module analysis showed that 8 out of 10 hub genes participated in Module 1 network and more than 70% genes of Module 2 consisted of collagen family members. Notably, transcription factors (TFs) regulatory network analysis indicated that E2F7, FOXM1, and NFYB were master regulators of Module 1, while CREB3L1 was the master regulator of Module 2. Experimental validation showed that CREB3L1, E2F7, and FOXM1 were significantly upregulated in ATC tissue and cell line when compared with normal thyroid group. In conclusion, the TFs regulatory network provided a more detail molecular mechanism underlying ATC occurrence and progression. TFs including E2F7, FOXM1, CREB3L1, and NFYB were likely to be master regulators of ATC progression, suggesting their potential role as molecular therapeutic targets in ATC treatment.