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In this study, we evaluated the biological role of miRNA-31-5p in papillary thyroid cancer (PTC).By using the real-time PCR, we measured miRNA-31-5p expression levels in 25 PTC tissues and in two human PTC cell lines (K1 and TPC-1). Then, K1 cells were transiently transfected with mirVana inhibitor or mirVana mimic to miRNA-31-5-p. Cell proliferation was determined by MTT and colony formation assays. The in vitro metastatic ability of thyroid cancer cells was evaluated by adhesion, migration and invasion assays. Epithelial mesenchymal transition (EMT) and Hippo pathway related gene and protein levels were evaluated by using the TaqMan™ Gene Expression Assays and western blot analysis, respectively.We found a significant increase of miR-31-5-p expression in tumor tissue and in K1 cells harboring the BRAF p.V600E mutation. Knockdown of miR-31-5p determined a reduction of cell proliferation, associated with a significant decrease in cell adhesion, migration and invasion properties. A downregulation of EMT markers and YAP/β-catenin axis was also observed.Our findings suggest that miRNA-31-5p acts as oncogenic miRNA in human thyrocytes and its overexpression may be involved in the BRAF-related tumorigenesis in PTCs, providing new understanding into its pathological role in PTC progression and invasiveness.

In the search for novel effective compounds to use in thyroid cancer (TC) unresponsive to current treatment, attention has recently focused on plant-derived compounds with anticancer activity. In this review, we discuss the preclinical studies demonstrating phytochemical activity against thyroid cancer cells. In particular, we describe their antiproliferative properties or ability to re-induce iodine retention, thus supporting their potential use as single agents or adjuvants in radioiodine-resistant thyroid cancer treatment.

Mutations in TERT promoter have been detected in the more aggressive papillary thyroid cancers (PTCs). To elucidate the role of TERT as an eligible molecular target in these tumors, the expression of hTERT was analyzed in a series of PTCs and the effects of both pharmacological and RNA-interference-induced hTERT silencing were investigated in two human PTC cell lines (K1 and BCPAP). The expression levels of hTERT mRNA and protein were evaluated by real-time PCR and western blot assays, respectively. Effects of hTERT silencing on PTC cell lines were analyzed by MTT, migration and western blot assays. Pharmacological inhibition of hTERT was performed using two bromodomain and extra-terminal (BET) inhibitors, JQ1 and I-BET762. hTERT expression results increased in 20 out of 48 PTCs, including tumors either positive or negative for the presence of hTERT promoter and/or BRAF mutations. In K1 and BCPAP cells, hTERT silencing determined a reduction in cell viability (~50% for K1 and ~70%, for BCPAP, vs control) and migration properties that were associated with a decrease of AKT phosphorylation and β-Catenin expression. Moreover, hTERT mRNA levels were down-regulated by two BET inhibitors, JQ1 and I-BET762, which at the same dosage (0.5 and 5 µM) reduced the growth of these thyroid cancer cells. These findings demonstrate that hTERT may represent an excellent therapeutic target in subgroups of aggressive PTCs.

A substantial proportion of patients with advanced thyroid carcinoma fail to respond to or at some point become refractory to conventional therapies. This resistance and the phenomena of thyroid cancer progression and metastasis themselves are thought to be related to tumor-cell sub-populations with stem-like properties. We isolated thyrospheres from four advanced thyroid carcinomas that were resistant to radioiodine therapy and analyzed their molecular profiles. ALDH activity and proteomic profile of main stem cell markers were used to assess stem cell properties. The TaqMan Low Density Array approach was used to evaluate the expression of several genes involved in the EMT process. The phosphorylation status of tyrosine kinase receptors (RTKs) was analyzed to identify potential markers for targeted therapies. We then investigated the effects of the EMT-inhibitor crizotinib on both cell proliferation and phosphorylation status of RTK targets. The cancer stem-like properties of a subset of cells from primary cultures of each tumor were demonstrated. A wide variability among thyrospheres arising from the four thyroid cancers in terms of ALDH activity, stem cell marker expression, and phosphoproteome profiling was present. Dysregulated expression of genes involved in the EMT was observed in all four thyrosphere lines. Treatment with crizotinib was ineffective in cancer stem-like cells, suggesting the presence of a mechanism of resistance in thyrospheres. Collectively, our data indicate that thyroid cancer stem-like populations vary markedly from tumor to tumor and require detailed molecular and biological characterization if they are to be used as the basis of "personalized" treatment of aggressive disease.

Recent studies have revealed in normal thyroid tissue the presence of the transcript of several phosphodiesterases (PDEs), enzymes responsible for the hydrolysis of cyclic nucleotides. In this work, we analyzed the expression of PDE5 in a series of human papillary thyroid carcinomas (PTCs) presenting or not BRAF V600E mutation and classified according to ATA risk criteria. Furthermore, we tested the effects of two PDE5 inhibitors (sildenafil, tadalafil) against human thyroid cancer cells. PDE5 gene and protein expression were analyzed in two different cohorts of PTCs by real-time PCR using a TaqMan micro-fluid card system and Western blot. MTT and migration assay were used to evaluate the effects of PDE5 inhibitors on proliferation and migration of TPC-1, BCPAP, and 8505C cells. In a first series of 36 PTCs, we found higher expression levels of PDE5A in tumors versus non-tumor (normal) tissues. PTCs with BRAF mutation showed higher levels of mRNA compared with those without mutation. No significant differences were detected between subgroups with low and intermediate ATA risk. Upregulation of PDE5 was also detected in tumor tissue proteins. Similar results were obtained analyzing the second cohort of 50 PTCs. Moreover, all tumor tissues with high PDE5 levels showed reduction of Thyroglobulin, TSH receptor, Thyroperoxidase, and NIS transcripts. In thyroid cancer cells in vitro, sildenafil and tadalafil determined a reduction of proliferation and cellular migration. Our findings demonstrate for the first time an overexpression of PDE5 in PTCs, and the ability of PDE5 inhibitors to block the proliferation of thyroid cancer cells in culture, therefore, suggesting that specific inhibition of PDE5 may be proposed for the treatment of these tumors.

The Hippo signal transduction pathway plays a crucial role in the control of cellular proliferation, so that its deregulation is believed to be involved in neoplastic transformation [1, 2]. A wide array of human cancers show a down-regulation of Hippo pathway, resulting in the activation of the co-transcription factors Yes-associated protein (YAP1) as well as the transcriptional coactivator with PDZ-binding motif (TAZ). In turn, the YAP/TAZ complex increases transcription of target proteins with oncogenic activity [1, 2]. Thus, targeting YAP has been tested to arrest cancer cell proliferation [3, 4].

Efficient treatment of radio refractory thyroid cancer is still a major challenge. The recent identification of genetic and epigenetic alterations present in almost all differentiated tumors has revealed novel molecular targets, which can hopefully be exploited to create new treatments for these tumors. This review looks briefly at some of the innovative strategies currently being investigated for the treatment the radioiodine-resistant thyroid cancers.

Somatic gene copy number variation contributes to tumor progression. Using comparative genomic hybridization (CGH) array, the presence of genomic imbalances was evaluated in a series of 27 papillary thyroid carcinomas (PTCs). To detect only somatic imbalances, for each sample, the reference DNA was from normal thyroid tissue of the same patient. The presence of the BRAF V600E mutation was also evaluated. Both amplifications and deletions showed an uneven distribution along the entire PTC cohort; amplifications were more frequent than deletions (mean values of 17.5 and 7.2, respectively). Number of aberration events was not even among samples, the majority of them occurring only in a small fraction of PTCs. Most frequent amplifications were detected at regions 2q35, 4q26, and 4q34.1, containing FN1, PDE5A, and GALNTL6 genes, respectively. Most frequent deletions occurred at regions 6q25.2, containing OPMR1 and IPCEF1 genes and 7q14.2, containing AOAH and ELMO1 genes. Amplification of FN1 and PDE5A genomic regions was confirmed by quantitative PCR. Frequency of amplifications and deletions was in relationship with clinical features and BRAF mutation status of tumor. In fact, according to the American Joint Committee on Cancer stage and American Thyroid Association (ATA) risk classification, amplifications are more frequent in higher risk samples, while deletions tend to prevail in the lower risk tumors. Analysis of single aberrations according to the ATA risk grouping shows that amplifications containing PDE5A, GALNTL6, DHRS3, and DOCK9 genes are significantly more frequent in the intermediate/high risk group than in the low risk group. Thus, our data would indicate that analysis of somatic genome aberrations by CGH array can be useful to identify additional prognostic variables.

IntroductionA causative role for activating mutations of the TSHreceptor (TSHR) has been demonstrated in the majority ofthyroid hyperfunctioning nodules, as well as for thefamilial and sporadic congenital nonautoimmune hyper-thyroidism [1, 2] and also the rare hyperfunctioning car-cinomas [3, 4]. More than 30 different amino acidsubstitutions have been described so far, and their char-acterization, together with in vitro mutagenesis studieshave been exploited for a structure–function analysis of thereceptor, leading to a structural model in which the areasinvolved in the binding to TSH or signal transduction havebeen depicted [5]. The emerging picture suggests theexistence of a molecular constraint which limits the con-stitutive activity of the receptor, removed by the interactionof TSH with the extracellular (EX) portion, but with animportant role played also by the transmembrane regions inthe dimerization process and the functional activation ofthe receptor [5, 6]. Herein we describe the discovery of twounusual heterozygous mutations occurring in two of seventhyroid functioning nodules detected as single nodules or ina multinodular goiter.Materials and methodsSpecimen of nodular tissues were collected and immedi-ately frozen after surgical removal from seven patients withsingle or multinodular hyperfunctioning goiter. Non-nod-ular normal tissue from the contralateral lobe was alsocollected. After RNA extraction and cDNA synthesis, PCRamplification of the exons 9 and 10 of the TSHR gene wasperformed as previously described [3, 4] and the amplifiedproducts sequenced with a BigDye Terminator version 3.1Cycle Sequencing kit in an automated 3130xl analyzer(both from Applied Biosystems, Foster City, CA, USA).The study protocol was approved by the local ethicscommittee.ResultsSequencing of TSH receptor gene in a small cohort ofseven patients with thyroid hyperfunctioning nodulesrevealed the presence, in two cases, of unusual somaticheterozygous mutations. The first was a deletion of D403

The presence of the sodium/iodide symporter (NIS) is the prerequisite for the use of the radioiodine in the treatment of thyroid cancer. Thus, stimulators of NIS expression and function are currently investigated in cellular models of various human malignancies, also including extrathyroid cancers. In this study, we analyzed the effects of the histone deacetylase inhibitors (HDACi), suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA), on NIS expression and function in rat Leydig testicular carcinoma cells (LC540). LC540 cells were exposed to SAHA 3 μM and VPA 3 mM (alone and in combination), and cell viability evaluated by MTT assay and cell counting, NIS mRNA and protein levels by using, respectively, real-time RT-PCR and western blotting. NIS function was evaluated by iodide uptake assay. We found that both HDACi were able to stimulate the transcription of NIS gene, but not its protein expression, while the association of SAHA and VPA increased both NIS transcript and protein levels, resulting in significant sixfold enhancement of radioiodine uptake capacity of LC540 cells. These data demonstrate the presence of an epigenetic control of NIS expression in Leydig tumor cells, suggesting the possibility to use the combination of these two HDACi for a radioiodine-based treatment of these malignancies.