Papillary Thyroid Carcinoma Samples Research Articles

MicroRNA-221 promotes papillary thyroid carcinoma cell migration and invasion via targeting RECK and regulating epithelial-mesenchymal transition.

AimThe aim of this study was to detect the effects and potential mechanisms of microRNA-221 on a series of biological behaviors of papillary thyroid carcinoma (PTC) cells in vitro and in vivo.MethodsFirst, we analyzed the relationship between the expression of miR-221 and several clinicopathological features of PTC patients and then detected the expression of the miR-221 in tumor tissues and cell lines. The effects of miR-221 on proliferation and invasion of PTC cells were verified by cell counting kit-8 (CCK-8) assay, wound healing assay and transwell assay. Western blot assay was applied to explore the correlation between miR-221 and RECK expression in PTC K1 cells. Finally, a xenograft model was established to further confirm the tumor-promoting effects of miR-221 in vivo.ResultsOur data indicated that miR-221 was relatively upregulated in metastatic PTC tissues. MiR-221 promoted the proliferation, migration and invasion activities of PTC K1 cells, following variations of epithelial–mesenchymal transition (EMT)-related protein expression. We identified RECK as a direct target of miR-221, revealed its expression to be inversely correlated with miR-221 in PTC samples and showed that its reintroduction reverses miR-221-induced PTC invasiveness. In addition, miR-221 was also verified to promote tumor growth and increase tumor volume and weight in vivo. Taken together, miR-221/RECK axis could be an effective way to regulate biological behaviors of PTC.ConclusionMiR-221 may be involved in PTC cell invasion and metastasis by targeting RECK, indicating that the miR-221/RECK pathway could be studied further as a potential new diagnostic or prognostic biomarker for PTC.

STAT3-induced upregulation of lncRNA ABHD11-AS1 promotes tumour progression in papillary thyroid carcinoma by regulating miR-1301-3p/STAT3 axis and PI3K/AKT signalling pathway.

ObjectivesEmerging evidences indicated the importance of long non‐coding RNAs (lncRNAs) in the tumorigenesis and deterioration of malignant tumours. To our knowledge, the study about lncRNAs in papillary thyroid carcinoma (PTC) is still inadequate. ABHD11‐AS1 was highly expressed in the PTC samples of The Cancer Genome Atlas database. This study focused on the biological function and mechanism of lncRNA ABHD11‐AS1 in PTC.Materials and methodsqRT‐PCR analysis was used to examine the expression of ABHD11‐AS1 in PTC tissues and cell lines. The prognostic significance of ABHD11‐AS1 for the patients with PTC was analysed with Kaplan‐Meier analysis. The effects of ABHD11‐AS1 knockdown on the cell proliferation and metastasis were evaluated by in vitro functional assays and in vivo experiments. The molecular mechanism which contributed to the oncogenic role of ABHD11‐AS1 in PTC was explored by conducting mechanism experiments. Rescue assays were carried out for final demonstration.ResultsHigh expression of ABHD11‐AS1 predicted poor prognosis for patients with PTC and promoted cell proliferation and metastasis in vitro and in vivo. ABHD11‐AS1 was activated by the transcription factor STAT3. ABHD11‐AS1 positively regulated PI3K/AKT signalling pathway. ABHD11‐AS1 acted as a competitive endogenous (ce) RNA to upregulate STAT3 by sponging miR‐1301‐3p.ConclusionsSTAT3‐induced lncRNA ABHD11‐AS1 promoted PTC progression by regulating PI3K/AKT signalling pathway and miR‐1301‐3p/STAT3 axis.

Long noncoding RNA cancer susceptibility candidate 2 suppresses papillary thyroid carcinoma growth by inactivating the AKT/ERK1/2 signaling pathway.

Cancer susceptibility candidate 2 (CASC2) and long noncoding RNA (lncRNA) have been identified asa tumor suppressor in colorectal, lung, renal, and stomach cancer as well as in patient gliomas, but the function of CASC2 in papillary thyroid carcinoma (PTC) is not yet clear. The present study aimed to explore the effects of CASC2 in PTC. The CASC2 expression was measured in PTC samples and normal tissues by using quantitative real-time polymerase chain reaction. The lentiviral vectors were used to establish CASC2 overexpression models in PTC cell lines to determine the effects of CASC2 on cell proliferation, apoptosis, migration, and invasion. A tumor xenograft animal model was used to examine the functions of overexpression CASC2. CASC2 expression was significantly decreased in PTC tumor tissues than adjacent normal tissues. CASC2 downregulation in PTC tissues significantly correlated with the tumor size, the presence of multifocal lesions, and the advanced pathological stage. CASC2 overexpression suppressed the cell proliferation and promoted apoptosis in PTC cell lines and CASC2 overexpression resulted in the inactivation of protein kinase B (PKB/AKT) and extracellular signal-regulated kinases (ERK1/2). The regulatory effects of CASC2 on PTC cell biological behavior were further enhanced by mitogen-activated protein kinase kinase (MEK) inhibitor U0126 or AKT1/2/3 inhibitor MK-2206 2HCl. CASC2 overexpression suppressed tumor growth in PTC cells in xenograft mouse models. Our results indicated that CASC2 significantly suppressed tumorigenesis in PTC and CASC2 may serve as a novel prognostic marker or therapeutic target.

MicroRNA‑509 targets PAX6 to inhibit cell proliferation and invasion in papillary thyroid carcinoma.

MicroRNAs (miRNAs/miRs) negatively regulate the expression of numerous genes and therefore contribute to the occurrence and development of papillary thyroid carcinoma (PTC). Hence, further investigation into the specific roles of miRNAs in PTC is valuable for developing effective therapeutic methods for patients with this disease. MiRNA‑509 is dysregulated and serves pivotal roles in several types of human cancer; however, the expression and roles of miR‑509 in PTC and its underlying mechanism require further investigation. In the present study, the expression of miR‑509 in PTC tissues and cell lines was detected and the specific functions of miR‑509 in the progression of PTC were investigated. Additionally, the molecular mechanisms underlying the action of miR‑509 in PTC were determined. The present study demonstrated that miR‑509 was significantly downregulated in PTC tissues and cell lines. MiR‑509 upregulation inhibited the PTC cell proliferation and invasion. Mechanistically, paired box 6 (PAX6) was identified as a novel target of miR‑509 in PTC cells. In clinical PTC samples, miR‑509 was significantly overexpressed and inversely correlated with PAX6 expression. PAX6 restoration effectively reversed the inhibitory effects of miR‑509 overexpression on PTC cell proliferation and invasion. These results demonstrated that miR‑509 may act as a tumor suppressor in PTC by directly targeting PAX6. Thus, miR‑509 may be a potential therapeutic target for the treatment of patients with PTC.

MMP-9-1562 C/T single nucleotide polymorphism associates with increased MMP-9 level and activity during papillary thyroid carcinoma progression

Papillary thyroid carcinoma (PTC), a common form of thyroid malignancy, displays significant variations in clinical features and outcome. The malignant transformation of the thyroid is driven by altered expression of many matrix-modulating enzymes, including matrix metalloproteinase-9 (MMP-9). A single nucleotide polymorphism in its promotor (-1562 C/T) is suspected to cause overexpression of MMP-9, which in turn contributes to development of a tumour unfavourable phenotype. The aim of this study was to investigate the impact of MMP-9 promotor genotype on MMP-9 expression in PTC samples, and to assess its value as a possible risk factor for developing PTC or its aggressive phenotype. A total of 105 PTC patients and 43 healthy controls were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. In order to estimate MMP-9 expression, PTC tissue sections were stained immunohistochemically. Statistical analysis showed that PTC cases and controls did not differ significantly in genotype frequencies (OR= 2.27, CI= 0.854-6.022). In PTC samples, the presence of the T allele was accompanied by elevated MMP-9 expression (p= 0.047) as well as a higher risk of developing extrathyroid extensions (p= 0.037) and high TNM stages (p= 0.009). Moreover, we observed overexpression of MMP-9 in cases presenting with extrathyroid invasion (p= 0.001), lymph node metastasis (p= 0.028), large tumour size (p= 0.031) and advanced stage (p= 0.005) compared to indolent tumours, along with enhanced enzymatic activity demonstrated by in situ zymography. Data suggests that MMP-9 (-1562 C/T) does not facilitate predisposition for PTC but affects the disease course by modulating MMP-9 expression. Genotyping MMP-9 provides important information which may prove beneficial in risk stratification of PTC patients.

Rapid detection of papillary thyroid carcinoma by fluorescence imaging using a \u03b3-glutamyltranspeptidase-specific probe: a pilot study

BackgroundNodular lesions of the thyroid gland, including papillary thyroid carcinoma (PTC), may be difficult to diagnose by imaging, such as in ultrasonic echo testing, or by needle biopsy. Definitive diagnosis is made by pathological examination but takes several days. A more rapid and simple method to clarify whether thyroid nodular lesions are benign or malignant is needed. Fluorescence imaging with γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) uses γ-glutamyltranspeptidase (GGT), a cell-surface enzyme, to hydrolyze the γ-glutamyl peptide and transfer the γ-glutamyl group. GGT is overexpressed in several cancers, such as breast, lung, and liver cancers. This imaging method is rapid and useful for detecting such cancers. In this study, we tried to develop a rapid fluorescence detection method for clinical samples of thyroid cancer, especially papillary carcinoma.MethodsFluorescence imaging with gGlu-HMRG was performed to detect PTC using 23 surgically resected clinical samples. A portable imaging device conveniently captured white-light images and fluorescence images with blue excitation light. Hematoxylin-eosin (HE) staining was used to evaluate which fluorescent regions coincided with cancer, and immunohistochemical examination was used to detect GGT expression.ResultsAll 16 PTC samples exhibited fluorescence after topical application of gGlu-HMRG, whereas the normal sections of each sample showed no fluorescence. HE staining revealed that each fluorescent region corresponded to a region with carcinoma. The PTC samples also exhibited GGT expression, as confirmed by immunohistochemistry.ConclusionsAll PTC samples were detected by fluorescence imaging with gGlu-HMRG. Thus, fluorescence imaging with gGlu-HMRG is a rapid, simple, and powerful detection tool for PTC.

Serum-based metabolic alterations in patients with papillary thyroid carcinoma unveiled by non-targeted 1H-NMR metabolomics approach.

Objective(s):As the most prevalent endocrine system malignancy, papillary thyroid carcinoma had a very fast rising incidence in recent years for unknown reasons besides the fact that the current methods in thyroid cancer diagnosis still hold some limitations. Therefore, the aim of this study was to improve the potential molecular markers for diagnosis of benign and malignant thyroid nodules to prevent unnecessary surgeries for benign tumors. Materials and Methods: In this study, 1H-NMR metabolomics platform was used to seek the discriminating serum metabolites in malignant papillary thyroid carcinoma (PTC) compared to benign multinodular goiter (MNG) and healthy subjects and also to better understand the disease mechanisms using bioinformatics analysis. Multivariate statistical analysis showed that PTC and MNG samples could be successfully discriminated in PCA and OPLS-DA score plots. Results:Significant metabolites that differentiated malignant and benign thyroid lesions included citrate, acetylcarnitine, glutamine, homoserine, glutathione, kynurenine, nicotinic acid, hippurate, tyrosine, tryptophan, β-alanine, and xanthine. The significant metabolites in the PTC group compared to healthy subjects also included scyllo- and myo-inositol, tryptophan, propionate, lactate, homocysteine, 3-methyl glutaric acid, asparagine, aspartate, choline, and acetamide. The metabolite sets enrichment analysis demonstrated that aspartate metabolism and urea cycle were the most important pathways in papillary thyroid cancer progression. Conclusion:The study results demonstrated that serum metabolic fingerprinting could serve as a viable method for differentiating various thyroid lesions and for proposing novel potential markers for thyroid cancers. Obviously, further studies are needed for the validation of the results.

Profile and clinical implication of circular RNAs in human papillary thyroid carcinoma.

BackgroundDifferently expressed circular RNAs (circRNAs) have been reported to play a considerable role in tumor behavior; however, the expression profile and biological function of circRNAs in papillary thyroid carcinoma (PTC) remains unknown. Thus, the study was aimed to characterize the circRNA expression profile to comprehensively understand the biological behavior of PTC.MethodsWe investigated the expression profile of circRNAs using circRNA microarray in three pairs of PTC and adjacent normal tissues. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to validate eight candidate circRNAs in 40 paired PTC tumors and adjacent normal samples. Next, we employed a bioinformatics tool to identify putative miRNA and circRNA-associated downstream genes, followed by constructing a network map of circRNA-miRNA-mRNA interactions and exploring the potential role of the candidate circRNAs.ResultsIn total, 206 up- and 177 downregulated circRNAs were identified in PTC tissues (fold change >1.5; P < 0.05). The expression levels of eight candidate circRNAs confirmed by qRT-PCR were significantly different between the PTC and normal samples. The downstream genes of candidate circRNAs participated in various biological processes and signaling pathways. The most up and downregulated circRNAs were hsa_circRNA_007148 and hsa_circRNA_047771. The lower expression level of hsa_circRNA_047771 was associated BRAFV600 mutation, lymph node metastasis (LNM), as well as with advanced TNM stage (all P < 0.05). The higher expression level of hsa_circRNA_007148 was significantly correlated with LNM (P < 0.05). The areas under receiver operating curve were 0.876 (95% CI [0.78–0.94]) for hsa_circRNA_047771 and 0.846 (95% CI [0.75–0.96]) for hsa_circRNA_007148.DiscussionThe study suggests that dysregulated circRNAs play a critical role in PTC pathogenesis. PTC-related hsa_circRNA_047771 and hsa_circRNA_007148 may serve as potential diagnostic biomarkers and prognostic predictors for PTC patients.

AP-2α expression in papillary thyroid carcinoma predicts tumor progression and poor prognosis.

BackgroundThe activator protein (AP)-2α is involved in a wide variety of biologic processes in tumor. However, little is known about the role of AP-2α in human papillary thyroid carcinoma (PTC).MethodsThe immunohistochemical method was used to detect AP-2α expression in 63 PTC cases. Western blotting was carried out to assess the change in expression of certain proteins. The bioinformatics analysis of 496 PTC samples comes from The Cancer Genome Atlas (TCGA). The Gene Set Enrichment Analysis (GSEA) was performed using TCGA data set. Cell transfection was used to induce related protein expression or to repress it by RNA interference procedures.ResultsOur results demonstrated that AP-2α expression was higher in tumor tissues than the corresponding adjacent nontumor tissues, the positive substances of AP-2α were observed mainly in the cytoplasm of PTC, and AP-2α was positively correlated with histologic type (P=0.026) of PTC patients. The high expression of AP-2α mRNA was associated significantly with tumor stages (P=0.011), histologic type (P=0.019), and independently predicted shorter overall survival (P=0.005) based on TCGA analysis. Patients with high AP-2α mRNA expression have shorter overall survival compared to those with low AP-2α mRNA expression, particularly in advanced tumor stages (III and IV) of PTC patients (P=0.011). Multivariate analysis suggested that AP-2α mRNA expression might be an independent prognostic indicator for the survival of patients with PTC (P=0.037). Moreover, the association between enhanced AP-2α expression and two pathways (notch signaling and focal adhesion) was revealed by GSEA, and then confirmed by cellular experiments.ConclusionTaken together, our findings suggest that AP-2α may be a potential prognostic molecular marker and therapeutic target for PTC patients.

Immunohistochemistry is a feasible method to screen BRAF V600E mutation in colorectal and papillary thyroid carcinoma

AimsTo investigate whether immunohistochemistry (IHC) could be used to screen BRAF mutation status in formalin-fixed paraffin-embedded (FFPE) colorectal carcinoma (CRC) and papillary thyroid carcinoma (PTC) samples. MethodsEight surgical resected samples, including 2 CRCs with mutated BRAF V600E, 2 PTCs with mutated BRAF V600E, 2 CRCs with wild-type BRAF and 2 PTCs with wild-type BRAF, were selected to explore the optimized IHC conditions for BRAF V600E (VE1) immunostaining using BenchmarkXT automated immunostainer VENTANA Medical System. BRAF V600E status was tested by optimized IHC and ARMS-PCR methods in 255 samples (123 CRCs and 132 PTCs). Sanger sequencing was performed to validate the BRAF V600E status if discordant results were found between optimized IHC and ARMS-PCR methods. ResultsAntigen retrieval time in 32 min and 64 min showed satisfactory intensity and homogeneity of BRAF V600E staining in CRC and PTC samples, respectively. The concordance between IHC and ARMS/Sanger sequencing was 99.2% (122/123) in CRCs and 96.2% (127/132) in PTCs. In CRCs, the sensitivity of IHC staining for BRAF V600E was 100% (3/3) and the specificity was 99.1% (119/120). In PTCs, the sensitivity was 100% (106/106) and specificity was 80.8% (21/26). The overall concordance across all cases was 97.6% (249/255). ConclusionThe appropriate antigen retrieval protocol is critical for accurate analysis of BRAF V600E status by Benchmark XT automated immunostainer. IHC is a suitable method to screen BRAF V600E mutation in FFPE samples of CRCs and PTCs.

Anaplastic lymphoma kinase (ALK) gene rearrangements in radiation‐related human papillary thyroid carcinoma after the Chernobyl accident

Childhood radiation exposure has been associated with increased papillary thyroid carcinoma (PTC) risk. The role of anaplastic lymphoma kinase (ALK) gene rearrangements in radiation‐related PTC remains unclear, but STRN‐ALK fusions have recently been detected in PTCs from radiation exposed persons after Chernobyl using targeted next‐generation sequencing and RNA‐seq. We investigated ALK and RET gene rearrangements as well as known driver point mutations in PTC tumours from 77 radiation‐exposed patients (mean age at surgery 22.4 years) and PTC tumours from 19 non‐exposed individuals after the Chernobyl accident. ALK rearrangements were detected by fluorescence in situ hybridisation (FISH) and confirmed with immunohistochemistry (IHC); point mutations in the BRAF and RAS genes were detected by DNA pyrosequencing. Among the 77 tumours from exposed persons, we identified 7 ALK rearrangements and none in the unexposed group. When combining ALK and RET rearrangements, we found 24 in the exposed (31.2%) compared to two (10.5%) in the unexposed group. Odds ratios increased significantly in a dose‐dependent manner up to 6.2 (95%CI: 1.1, 34.7; p = 0.039) at Iodine‐131 thyroid doses >500 mGy. In total, 27 cases carried point mutations of BRAF or RAS genes, yet logistic regression analysis failed to identify significant dose association. To our knowledge we are the first to describe ALK rearrangements in post‐Chernobyl PTC samples using routine methods such as FISH and IHC. Our findings further support the hypothesis that gene rearrangements, but not oncogenic driver mutations, are associated with ionising radiation‐related tumour risk. IHC may represent an effective method for ALK‐screening in PTCs with known radiation aetiology, which is of clinical value since oncogenic ALK activation might represent a valuable target for small molecule inhibitors.

Expression of BANCR promotes papillary thyroid cancer by targeting thyroid stimulating hormone receptor.

Papillary thyroid carcinoma (PTC) is the most common form of non-medullary thyroid cancer, accounting for ~80% of all cases of thyroid cancer. The aim of the present study was to explore the role of BRAF-activated long noncoding RNA (BANCR) in the development of PTC. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), the mRNA expression levels of BANCR, thyroid-stimulating hormone receptor (TSHR) and cyclin D1 between PTC and benign control thyroid nodule tissue samples from 60 patients were determined. Using RT-qPCR and western blot analysis, the expression levels of TSHR and cyclin D1 mRNA and protein were determined in cells transfected with BANCR-small interfering (si)RNA. An MTT assay and flow cytometry were used to analyze the effect of BANCR knockdown on the proliferation and cell cycle distribution of IHH-4 PTC cells. The expression of BANCR, TSHR and cyclin D1 was increased in the PTC group compared with the control group based on the RT-qPCR data. The transfection of IHH-4 cells with BANCR-siRNA induced the inhibition of TSHR and cyclin D1 expression compared with a transfection control. In addition, the proliferation of the IHH-4 cells transfected with BANCR-siRNA was suppressed, relative to the transfection control, and cells arrested in the G0/G1 phase, potentially due to the inhibition of the expression of cyclin D1. The data suggested that the expression of BANCR may promote the development of malignant thyroid nodules via the modulation of TSHR expression and its downstream effector, cyclin D1.

Competing endogenous RNA regulatory network in papillary thyroid carcinoma.

The present study aimed to screen all types of RNAs involved in the development of papillary thyroid carcinoma (PTC). RNA-sequencing data of PTC and normal samples were used for screening differentially expressed (DE) microRNAs (DE-miRNAs), long non-coding RNAs (DE-lncRNAs) and genes (DEGs). Subsequently, lncRNA-miRNA, miRNA-gene (that is, miRNA-mRNA) and gene-gene interaction pairs were extracted and used to construct regulatory networks. Feature genes in the miRNA-mRNA network were identified by topological analysis and recursive feature elimination analysis. A support vector machine (SVM) classifier was built using 15 feature genes, and its classification effect was validated using two microarray data sets that were downloaded from the Gene Expression Omnibus (GEO) database. In addition, Gene Ontology function and Kyoto Encyclopedia Genes and Genomes pathway enrichment analyses were conducted for genes identified in the ceRNA network. A total of 506 samples, including 447 tumor samples and 59 normal samples, were obtained from The Cancer Genome Atlas (TCGA); 16 DE-lncRNAs, 917 DEGs and 30 DE-miRNAs were screened. The miRNA-mRNA regulatory network comprised 353 nodes and 577 interactions. From these data, 15 feature genes with high predictive precision (>95%) were extracted from the network and were used to form an SVM classifier with an accuracy of 96.05% (486/506) for PTC samples downloaded from TCGA, and accuracies of 96.81 and 98.46% for GEO downloaded data sets. The ceRNA regulatory network comprised 596 lines (or interactions) and 365 nodes. Genes in the ceRNA network were significantly enriched in ‘neuron development’, ‘differentiation’, ‘neuroactive ligand-receptor interaction’, ‘metabolism of xenobiotics by cytochrome P450’, ‘drug metabolism’ and ‘cytokine-cytokine receptor interaction’ pathways. Hox transcript antisense RNA, miRNA-206 and kallikrein-related peptidase 10 were nodes in the ceRNA regulatory network of the selected feature gene, and they may serve import roles in the development of PTC.

The mitochondrial DNA control region might have useful diagnostic and prognostic biomarkers for thyroid tumors

Background: It is currently present in the literature that mitochondrial DNA (mtDNA) defects are associated with a great number of diseases including cancers. The role of mitochondrial DNA (mtDNA) variations in the development of thyroid cancer is a highly controversial topic. In this study, we investigated the role of mt-DNA control region (CR) variations in thyroid tumor progression and the influence of mtDNA haplogroups on susceptibility to thyroid tumors. Material & method: For this purpose, totally 108 hot thyroid nodules (HTNs), 95 cold thyroid nodules (CTNs), 48 papillary thyroid carcinoma (PTC) samples with their surrounding tissues and 104 healthy control subject9s blood samples were screened for entire mtDNA CR variations by using Sanger sequencing. The obtained DNA sequences were anaysed with the mistomaster, a web-based bioinformatics tool. Results: MtDNA haplogroup U was significantly associated with susceptibility to benign and malign thyroid entities on the other hand J haplogroup was associated with a protective role for benign thyroid nodules. Besides, 8 SNPs (T146C, G185A, C194T, C295T, G16129A, T16304C, A16343G and T16362C) in mtDNA CR region were associated with the occurrence of benign and malign thyroid nodules in Turkish population. By contrast with the healthy Turkish population and HTNs, frequency of C7 repeats in D310 polycytosine sequence was found higher in cold thyroid nodules and PTC samples. Beside this, the frequency of somatic mutations in mtMSI regions including T16189C and D514 CA dinucleotide repeats were found higher in PTC samples than the benign thyroid nodules. Conversely, the frequency of somatic mutations in D310 was detected higher in HTNs than CTNs and PTCs. Conclusion: mtDNA D310 instability do not play a role in tumorogenesis of the PTC but the results indicates that it might be used as a diagnostic clonal expansion biomarker for premalignant thyroid tumor cells. Beside this, D514 CA instability might be used as prognostic biomarker in PTCs. Also, we showed that somatic mutation rate is less frequent in more aggressive tumors when we examined micro- and macro carcinomas as well as BRAFV600E mutation.

NKX2.5 is expressed in papillary thyroid carcinomas and regulates differentiation in thyroid cells

BackgroundNKX2.5 is a transcription factor transiently expressed during thyroid organogenesis. Recently, several works have pointed out the oncogenic role of NKX2.5 in a variety of tumors. We therefore hypothesized that NKX2.5 could also play a role in thyroid cancer.MethodsThe validation of NKX2.5 expression was assessed by immunohistochemistry analysis in a Brazilian case series of 10 papillary thyroid carcinoma (PTC) patients. Then, the long-term prognostic value of NKX2.5 and its correlation with clinicopathologic features of 51 PTC patients was evaluated in a cohort with 10-years follow-up (1990–1999). Besides, the effect of NKX2.5 overexpression on thyroid differentiation markers and function was also investigated in a non-tumor thyroid cell line (PCCL3).ResultsNKX2.5 was shown to be expressed in most PTC samples (8/10, case series; 27/51, cohort). Patients who had tumors expressing NKX2.5 showed lower rates of persistence/recurrence (p = 0.013). Overexpression of NKX2.5 in PCCL3 cells led to: 1) downregulation of thyroid differentiation markers (thyrotropin receptor, thyroperoxidase and sodium-iodide symporter); 2) reduced iodide uptake; 3) increased extracellular H2O2 generation, dual oxidase 1 mRNA levels and activity of DuOx1 promoter.ConclusionsIn summary, NKX2.5 is expressed in most PTC samples analyzed and its presence correlates to better prognosis of PTC. In vitro, NKX2.5 overexpression reduces the expression of thyroid differentiation markers and increases ROS production. Thus, our data suggests that NKX2.5 could play a role in thyroid carcinogenesis.

Suppression of CCT3 inhibits malignant proliferation of human papillary thyroid carcinoma cell.

Papillary thyroid carcinoma (PTC) is the most frequently occurring subtype of thyroid cancer. A certain portion of PTCs can progress to recurrent metastatic cancer. Currently, there remains no effective molecular target therapy for PTCs. As a subunit of the chaperonin containing TCP1 (CCT) complex, CCT3 is involved in various biological processes. CCT3 has been reported to drive the proliferation of hepatocellular carcinoma cells. Nevertheless, it remains unknown whether CCT3 regulates the development of PTC. The present study examined CCT3 protein expression in 30 PTC samples from patients undergoing thyroidectomy. A significant increase was observed in CCT3 expression in the PTC samples compared with the matched adjacent normal thyroid tissues. Lentiviral-mediated small interfering RNAs were used to knock down CCT3 in K1 cells. It was observed that the expression of CCT3 was significantly suppressed in K1 cells infected with lentivirus containing a CCT3-targeting short hairpin RNA. Our results showed that CCT3 knockdown markedly decreased the proliferation and cell cycle progression of K1 cells. In addition, the knockdown of CCT3 induced apoptosis of K1 cell. Taken together, the findings of the present study indicated that CCT3 presents as a potential molecular marker of PTC and regulates the development of PTC in humans.

LIMD2 Is Overexpressed in BRAF V600E-Positive Papillary Thyroid Carcinomas and Matched Lymph Node Metastases.

We previously described that LIM domain containing 2 (LIMD2) overexpression was closely correlated with metastatic process in papillary thyroid carcinoma (PTC). We here evaluated the expression of LIMD2 in a series of non-metastatic and metastatic PTC and their matched lymph node metastases via immunohistochemistry. LIMD2 was expressed in 74 (81%) of primary PTC and 35 (95%) of lymph node metastases. Sub-analysis performed in 37 matched samples demonstrated that in four cases, LIMD2 is expressed in lymph node metastases, while it is not expressed in primary tumors. Moreover, in eight cases, the staining intensity of LIMD2 was stronger in the patient-matched lymph node metastases than in the primary tumors. Next, the expression of LIMD2 was correlated with clinical pathological parameters and BRAF V600E and RET/PTC mutational status. The expression of LIMD2 in primary tumors was correlated with the presence of BRAF V600E mutation (P= 0.0338). Western blot analysis in thyroid cell lines demonstrated that LIMD2 is expressed in two PTC cell lines, while it is not expressed in normal thyroid and follicular thyroid carcinoma cell lines. Importantly, its expression was higher in a PTC cell line that harbors BRAF V600E mutation than in a PTC cell line that harbors RET/PTC1. The available genomic profiling data generated by The Cancer Genome Atlas Research Network confirmed that LIMD2 expression is higher in BRAF-like PTC samples. Our data suggest that LIMD2 may play an important role in the metastatic process of PTC, predominantly in BRAF V600E-positive tumors.

Identification of a novel HRAS variant and its association with papillary thyroid carcinoma.

HRas proto-oncogene (HRAS) is one of the most commonly mutated genes in thyroid cancer, with mutations frequently occurring in the follicular and Hurthle cell subtypes. However, the contribution of mutations in HRAS to papillary thyroid carcinoma (PTC) progression and the tall-cell variant (TCV) is poorly understood. The aim of the present study was to investigate the somatic genetic variants present in HRAS in patients with PTC, and to investigate the association of these mutations with PTC. The present study is a retrospective case-control study using tumor samples collected from 139 patients with PTC and blood samples from 195 healthy individuals. All patient samples were screened for mutations in 'hotspot' regions of HRAS and B-raf proto-oncogene (BRAF) by single-stranded conformational polymorphism analysis, followed by direct sequencing. A novel variant (IVS1-82del gctgggcctggg) in the HRAS 5'-untranslated region was identified. There was no difference in age or sex of patients with PTC and the healthy controls; however, the HRAS variant was more frequently detected in PTC tissue than in the healthy control samples (37 vs. 26%, P=0.04). There was no association between the HRAS variant and age, sex, tumor size, encapsulation, multifocality/intra-thyroidal spread, Tumor-Node-Metastasis stage, history of Hashimoto's disease, BRAF V600E mutation or PTC subtype (all P>0.05). There were differences of BRAF V600E distribution among different subtypes (χ2=6.390, P=0.041). HRAS variant co-occurring with the BRAF V600E mutation accounted for 31.6% of the total number (P=0.196). Therefore, this novel variant of HRAS (IVS1-82del gctgggcctggg) may be associated with PTC; however, larger scale studies are required to assess the contribution of this novel HRAS variant to PTC progression.

Abstract A13: Gene body methylation could regulate the expression of genes associated to papillary thyroid carcinomas

Abstract Background: Papillary thyroid carcinoma (PTC) is the histological subtype more frequently reported among the endocrine malignancies. Deregulation of DNA methylation has been extensively reported in cancer with hypermethylation usually found in promoter and hypomethylation in nonpromoter regions. The methylation profile of PTC revealed altered genome regions; however, its impact on the gene expression remains poorly understood. DNA methylation profile in PTC patients was performed, aiming to investigate the role of novel genic and intergenic regions on the expression of genes. Patients and Methods: The methylation profile of 41 patients diagnosed with PTC was evaluated using the Methylation 450 Human Infinium®BeadChip platform (Illumina). The data were normalized and analyzed using SVA, watermelon, and LIMMA packages. Delta beta of 0.15 and adjusted p-value &amp;lt;0.05 were used to identify differentially methylated probes between PTC and non-neoplastic thyroid tissue (NT). An integrative analysis was performed using the expression data obtained in 34 matched-cases. The results were submitted to a cross-validation with The Cancer Genome Atlas (TCGA) database. An in silico analysis was performed using genes/probes showing negative correlation and confirmed by TCGA using the Ingenuity Pathways Analysis. ERBB3, FGF1, FGFR2, GABRB2, HMGA2, and RDH5 were validated by pyrosequencing and RT-qPCR methods. Results: Methylation analysis revealed 4,995 differentially methylated probes. A global hypomethylation was found in PTC samples (88% hypomethylated probes) mostly in nonpromoter regions (especially in gene body), distant from CGI and enriched by enhancers. The integrative analysis between gene expression and DNA methylation revealed 185 and 38 genes with negative and positive correlation, respectively. Genes showing negative correlation highlighted FGF and retinoic acid signaling as crucial pathways disrupted by DNA methylation in PTC. The selected genes were confirmed as altered in both methylation and expression levels. Interestingly, four of them (ERBB3, FGF1, GABRB2, and HMGA2) were mapped in gene body, suggesting that methylation in this region regulates the gene expression. Conclusion: Loss of methylation was extensively observed in PTC, especially in nonpromoter, poor CGI, and enhancer-enriched regions. Gene body and promoter regions have the potential to influence the gene expression levels (both repressing and inducing). Genes potentially regulated by DNA methylation were identified from the integrative and cross-validation analyses. ERBB3, FGF1, FGFR2, GABRB2, HMGA2, and RDH5 biomarkers are regulated by methylation in PTC samples. Citation Format: Caroline Moraes Beltrami, Mateus Camargo Barros-Filho, Mariana Bisarro dos Reis, Fábio Albuquerque Marchi, Hellen Kuasne, Skirant Ambatipudi, Zdenko Herceg, Luiz Paulo Kowalski, Silvia Regina Rogatto. Gene body methylation could regulate the expression of genes associated to papillary thyroid carcinomas [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A13.

Telomerase reverse transcriptase mutations are independent predictor of disease-free survival in Middle Eastern papillary thyroid cancer.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Tumor recurrence occurs in ∼20% of PTCs and some reach advanced stages. Promoter mutation in the telomerase reverse transcriptase (TERT) gene is identified to be a prognostic marker in PTC. However, the contribution of TERT promoter mutation to cancer progression in PTC patients is still not fully understood. In this study, we investigated the incidence of TERT promoter mutations and TERT protein expression and their association with clinicopathological outcomes in a large cohort of PTC samples using direct sequencing technology and immunohistochemistry. Furthermore, two PTC cell lines were utilized to investigate role of TERT mutations in mediating metastasis. Two promoter hotspot mutations C228T and C250T were identified in 18.0% (167/927) of our cohort and were significantly associated with poor 5 years disease-free survival and distant metastasis of PTC. TERT protein overexpression was noted in 20.1% of our PTC cohort and was significantly associated with poor prognostic markers such as older age, extrathyroidal extension and Stage IV tumors. A significant association was also found between TERT overexpression and epithelial-mesenchymal transition (EMT) markers. Functional analysis showed that TERT inhibition reduced cell growth, invasion, migration and angiogenesis in PTC via suppression of EMT in PTC cells. Our results suggest that TERT promoter mutation is an independent predictor of disease-free survival and might drive the metastasis, and downregulation of TERT could potentiate antitumor and antimetastatic activities in PTC.