Using CRISPR/Cas9 to Edit a Thyroid Cancer Cell Line.

Thyroid cancer is the most prevalent endocrine malignancy, comprising multiple types of cancer, with distinct clinical-pathological characteristics. The oncogenesis of thyroid cancer is related to genetic alterations in MAPK signaling that induce proliferation and modulate noncoding genes, such as microRNAs and long noncoding RNAs. In this context, CRISPR/Cas9 emerges as a potential tool to modify gene sequence and modulate gene expression in thyroid cancer cell lines. In this chapter, we explore some of the current studies in which researchers have applied CRISPR/Cas9 in vitro to investigate thyroid cancer biology (Fig. 5.1).

Modulation of EZH2 Activity Induces an Antitumoral Effect and Cell Redifferentiation in Anaplastic Thyroid Cancer.

Anaplastic thyroid cancer (ATC) is a rare and lethal form of thyroid cancer that requires urgent investigation of new molecular targets involved in its aggressive biology. In this context, the overactivation of Polycomb Repressive Complex 2/EZH2, which induces chromatin compaction, is frequently observed in aggressive solid tumors, making the EZH2 methyltransferase a potential target for treatment. However, the deregulation of chromatin accessibility is yet not fully investigated in thyroid cancer. In this study, EZH2 expression was modulated by CRISPR/Cas9-mediated gene editing and pharmacologically inhibited with EZH2 inhibitor EPZ6438 alone or in combination with the MAPK inhibitor U0126. The results showed that CRISPR/Cas9-induced EZH2 gene editing reduced cell growth, migration and invasion in vitro and resulted in a 90% reduction in tumor growth when EZH2-edited cells were injected into an immunocompromised mouse model. Immunohistochemistry analysis of the tumors revealed reduced tumor cell proliferation and less recruitment of cancer-associated fibroblasts in the EZH2-edited tumors compared to the control tumors. Moreover, EZH2 inhibition induced thyroid-differentiation genes' expression and mesenchymal-to-epithelial transition (MET) in ATC cells. Thus, this study shows that targeting EZH2 could be a promising neoadjuvant treatment for ATC, as it promotes antitumoral effects in vitro and in vivo and induces cell differentiation.

hnRNP A1 and hnRNP C associate with miR-17 and miR-18 in thyroid cancer cells.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are essential players in the regulation of gene expression. The majority of the twenty different hnRNP proteins act through the modulation of pre-mRNA splicing. Most have been shown to regulate the expression of critical genes for the progression of tumorigenic processes and were also observed to be overexpressed in several types of cancer. Moreover, these proteins were described as essential components for the maturation of some microRNAs (miRNAs). In the human genome, over 70% of miRNAs are transcribed from introns; therefore, we hypothesized that regulatory proteins involved with splicing could be important for their maturation. Increased expression of the miR-17-92 cluster has already been shown to be related to the development of many cancers, such as thyroid, lung, and lymphoma. In this article, we show that overexpression of hnRNP A1 and hnRNP C in BCPAP thyroid cancer cells directly affects the expression of miR-17-92 miRNAs. Both proteins associate with the 5'-end of this cluster, strongly precipitate miRNAs miR-17 and miR-18a and upregulate the expression of miR-92a. Upon overexpression of these hnRNPs, BCPAP cells also show increased proliferation, migration, and invasion rates, suggesting upregulation of these proteins and miRNAs is related to an enhanced tumorigenic phenotype.

Gene Editing with CRISPR/Cas Methodology and Thyroid Cancer: Where Are We?

Important advances on the role of genetic alterations in thyroid cancer have been achieved in the last two decades. One key reason is linked to the development of technical approaches that allowed for the mimicking of genetic alterations in vitro and in vivo and, more recently, the gene editing methodology. The CRISPR/Cas methodology has emerged as a tangible tool for editing virtually any DNA sequence in the genome. To induce a double-strand break and programmable gene editing, Cas9 endonuclease is guided by a single-guide RNA (sgRNA) that is complementary to the target sequence in DNA. The gene editing per se occurs as the cells repair the broken DNA and may erroneously change the original DNA sequence. In this review, we explore the principles of the CRISPR/Cas system to facilitate an understanding of the mainstream technique and its applications in gene editing. Furthermore, we explored new applications of CRISPR/Cas for gene modulation without changing the DNA sequence and provided a Dry Lab experience for those who are interested in starting "CRISPRing" any given gene. In the last section, we will discuss the progress in the knowledge of thyroid cancer biology fostered by the CRISPR/Cas gene editing tools.

Targeting the Highly Expressed microRNA miR-146b with CRISPR/Cas9n Gene Editing System in Thyroid Cancer.

Thyroid cancer is the most common endocrine malignancy, and the characterization of the genetic alterations in coding-genes that drive thyroid cancer are well consolidated in MAPK signaling. In the context of non-coding RNAs, microRNAs (miRNAs) are small non-coding RNAs that, when deregulated, cooperate to promote tumorigenesis by targeting mRNAs, many of which are proto-oncogenes and tumor suppressors. In thyroid cancer, miR-146b-5p is the most overexpressed miRNA associated with tumor aggressiveness and progression, while the antisense blocking of miR-146b-5p results in anti-tumoral effect. Therefore, inactivating miR-146b has been considered as a promising strategy in thyroid cancer therapy. Here, we applied the CRISPR/Cas9n editing system to target the MIR146B gene in an aggressive anaplastic thyroid cancer (ATC) cell line. For that, we designed two single-guide RNAs cloned into plasmids to direct Cas9 nickase (Cas9n) to the genomic region of the pre-mir-146b structure to target miR-146b-5p and miR-146b-3p sequences. In this plasmidial strategy, we cotransfected pSp-Cas9n-miR-146b-GuideA-puromycin and pSp-Cas9n-miR-146b-GuideB-GFP plasmids in KTC2 cells and selected the puromycin resistant + GFP positive clones (KTC2-Cl). As a result, we observed that the ATC cell line KTC2-Cl1 showed a 60% decrease in the expression of miR-146b-5p compared to the control, also showing reduced cell viability, migration, colony formation, and blockage of tumor development in immunocompromised mice. The analysis of the MIR146B edited sequence shows a 5 nt deletion in the miR-146b-5p region and a 1 nt deletion in the miR-146b-3p region in KTC2-Cl1. Thus, we developed an effective CRISPR/Cas9n system to edit the MIR146B miRNA gene and reduce miR-146b-5p expression which constitutes a potential molecular tool for the investigation of miRNAs function in thyroid cancer.

miR-200 deficiency promotes lung cancer metastasis by activating Notch signaling in cancer-associated fibroblasts.

Lung adenocarcinoma, the most prevalent lung cancer subtype, is characterized by its high propensity to metastasize. Despite the importance of metastasis in lung cancer mortality, its underlying cellular and molecular mechanisms remain largely elusive. Here, we identified miR-200 miRNAs as potent suppressors for lung adenocarcinoma metastasis. miR-200 expression is specifically repressed in mouse metastatic lung adenocarcinomas, and miR-200 decrease strongly correlates with poor patient survival. Consistently, deletion of mir-200c/141 in the KrasLSL-G12D/+ ; Trp53flox/flox lung adenocarcinoma mouse model significantly promoted metastasis, generating a desmoplastic tumor stroma highly reminiscent of metastatic human lung cancer. miR-200 deficiency in lung cancer cells promotes the proliferation and activation of adjacent cancer-associated fibroblasts (CAFs), which in turn elevates the metastatic potential of cancer cells. miR-200 regulates the functional interaction between cancer cells and CAFs, at least in part, by targeting Notch ligand Jagged1 and Jagged2 in cancer cells and inducing Notch activation in adjacent CAFs. Hence, the interaction between cancer cells and CAFs constitutes an essential mechanism to promote metastatic potential.

Age-related increase of let-7 family microRNA in rat retina and vitreous.

Vitreous alterations occur from early stages and continue through the normal aging, with gradual lamellae formation and the appearance of liquefied spaces, which eventually leads to complications, such as retinal tear, retinal detachment, and intravitreal hemorrhage. The aim of the present study was to investigate the expression of let-7 miRNA family in the vitreous and retina in newborn (1-3- day-old), young adult (2-month-old), and aging (12-month-old) rats, as well as their role as regulators of vitreous components. MicroRNAs are small, non-coding RNAs that post-transcriptionally regulate gene expression. Our results showed detection of all investigated let-7 isoforms (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f and let-7i) in the retina and vitreous. Although most let-7 members were significantly upregulated in the vitreous during development, only let-7b, let-7c, and let-7e followed this same expression pattern in the retina. Let-7b and -7c increased in aging vitreous as well, and were expressed in vitro by Müller glial cells and their extracellular vesicles. Moreover, let-7 targeted hyaluronan synthase 2 (Has2) mRNA, a synthesizing enzyme of hyaluronan. These observations indicate that let-7 function is important during retina and vitreous development, and that isoforms of let-7 increased with aging, potentially modulating hyaluronan content.

Genetic Mutations and Variants in the Susceptibility of Familial Non-Medullary Thyroid Cancer.

Thyroid cancer is the most frequent endocrine malignancy with the majority of cases derived from thyroid follicular cells and caused by sporadic mutations. However, when at least two or more first degree relatives present thyroid cancer, it is classified as familial non-medullary thyroid cancer (FNMTC) that may comprise 3-9% of all thyroid cancer. In this context, 5% of FNMTC are related to hereditary syndromes such as Cowden and Werner Syndromes, displaying specific genetic predisposition factors. On the other hand, the other 95% of cases are classified as non-syndromic FNMTC. Over the last 20 years, several candidate genes emerged in different studies of families worldwide. Nevertheless, the identification of a prevalent polymorphism or germinative mutation has not progressed in FNMTC. In this work, an overview of genetic alteration related to syndromic and non-syndromic FNMTC is presented.

Thyroid Follicular Cell Loss of Differentiation Induced by MicroRNA miR-17-92 Cluster Is Attenuated by CRISPR/Cas9n Gene Silencing in Anaplastic Thyroid Cancer.

Background: Loss of the expression of thyroid differentiation markers such as sodium iodide symporter (NIS) and, consequently, radioiodine refractoriness is observed in aggressive papillary thyroid cancer and anaplastic thyroid cancer (ATC) that may harbor the BRAFV600E mutation. Activation of the BRAFV600E oncogene in thyroid follicular cells induces the expression of the miR-17-92 cluster that comprises seven mature microRNAs (miRNAs). miRNAs are a class of endogenous small RNAs (∼22 nt) that regulate gene expression post-transcriptionally. Indeed, miR-17-92 is overexpressed in ATC, and in silico prediction shows the potential targeting of thyroid transcription factors and tumor suppressor pathways. In this study, we aimed to investigate the role of the miR-17-92 cluster in thyroid cell differentiation and function. Methods:miR-17-92 silencing was performed using CRISPR/Cas9n-guided genomic editing of the miR-17-92 gene in the KTC2 ATC cell line, and miR-17-92 cluster or individual miRNAs were overexpressed in PCCl3 thyroid cells to evaluate the influence in thyroid cell differentiation and cell function. Results: In this study, we demonstrate that CRISPR/Cas9n gene editing of the miR-17-92 cluster results in promotion of thyroid follicular cell differentiation (NIS, thyroperoxidase, thyroglobulin, PAX8, and NKX2-1 expression) in the KTC2 ATC cell line and inhibits cell migration and proliferation by restoring transforming growth factor beta (TGF-ß) signaling pathway responsiveness. Moreover, induction of the miR-17-92 cluster in normal thyroid follicular cells strongly impairs thyroid differentiation and induces a pro-oncogenic effect by blocking TGF-ß signaling and increasing cell migration. Conclusions:miR-17-92 is a potent regulator of thyroid follicular cell differentiation, and CRISPR/Cas9n-mediated editing of the miR-17-92 gene efficiently blocks miR-17-92 expression in the KTC2 ATC cell line, resulting in improvement of thyroid differentiation. Thus, targeting miR-17-92 could provide a potential molecular approach to restoring thyroid cell differentiation and NIS expression in aggressive thyroid cancer.

Interplay of TGFβ signaling and microRNA in thyroid cell loss of differentiation and cancer progression

ABSTRACT Thyroid cancer has been rapidly increasing in prevalence among humans in last 2 decades and is the most prevalent endocrine malignancy. Overall, thyroid-cancer patients have good rates of long-term survival, but a small percentage present poor outcome. Thyroid cancer aggressiveness is essentially related with thyroid follicular cell loss of differentiation and metastasis. The discovery of oncogenes that drive thyroid cancer (such as RET, RAS, and BRAF), and are aligned in the MAPK/ERK pathway has led to a new perspective of thyroid oncogenesis. The uncovering of additional oncogene-modulated signaling pathways revealed an intricate and active signaling cross-talk. Among these, microRNAs, which are a class of small, noncoding RNAs, expanded this cross-talk by modulating several components of the oncogenic network - thus establishing a new layer of regulation. In this context, TGFβ signaling plays an important role in cancer as a dual factor: it can exert an antimitogenic effect in normal thyroid follicular cells, and promote epithelial-to-mesenchymal transition, cell migration, and invasion in cancer cells. In this review, we explore how microRNAs influence the loss of thyroid differentiation and the increase in aggressiveness of thyroid cancers by regulating the dual function of TGFβ. This review provides directions for future research to encourage the development of new strategies and molecular approaches that can improve the treatment of aggressive thyroid cancer.

Interplay of TGFß signaling and microRNA in thyroid cell loss of differentiation and cancer progression.

Thyroid cancer has been rapidly increasing in prevalence among humans in last 2 decades and is the most prevalent endocrine malignancy. Overall, thyroid-cancer patients have good rates of long-term survival, but a small percentage present poor outcome. Thyroid cancer aggressiveness is essentially related with thyroid follicular cell loss of differentiation and metastasis. The discovery of oncogenes that drive thyroid cancer (such as RET, RAS, and BRAF), and are aligned in the MAPK/ERK pathway has led to a new perspective of thyroid oncogenesis. The uncovering of additional oncogene-modulated signaling pathways revealed an intricate and active signaling cross-talk. Among these, microRNAs, which are a class of small, noncoding RNAs, expanded this cross-talk by modulating several components of the oncogenic network - thus establishing a new layer of regulation. In this context, TGFß signaling plays an important role in cancer as a dual factor: it can exert an antimitogenic effect in normal thyroid follicular cells, and promote epithelial-to-mesenchymal transition, cell migration, and invasion in cancer cells. In this review, we explore how microRNAs influence the loss of thyroid differentiation and the increase in aggressiveness of thyroid cancers by regulating the dual function of TGFß. This review provides directions for future research to encourage the development of new strategies and molecular approaches that can improve the treatment of aggressive thyroid cancer.

The Highly Expressed FAM83F Protein in Papillary Thyroid Cancer Exerts a Pro-Oncogenic Role in Thyroid Follicular Cells.

Thyroid cancer is the most common endocrine cancer with predominant prevalence of papillary thyroid cancer (PTC) histotype. MAPK signaling genetic alterations are frequent in PTC, affecting more than 80% of cases. These alterations constitutively activate MAPK signaling cross-regulating different pro-oncogenic pathways. However, additional molecular alterations associated with thyroid cancer are not completely understood. In this extent, the new family of proteins named FAM83 (FAMily with sequence similarity 83) was recently identified as mediator of oncogenic signaling in different types of cancer. Here we report FAM83F as a novel highly expressed protein in PTC. We evaluated FAM83F levels in 106 PTC specimens, 34 goiter, and 41 adjacent non-tumoral human thyroid, and observed FAM83F cytoplasmic overexpression in 71% of PTC (76 of 106) while goiter tissues showed nuclear positivity and normal thyroid showed no staining by immunohistochemistry. Moreover, TSH-induced goiter and BRAF T1799A -induced PTC animal models also showed FAM83F activation. In vitro, we generated a stable thyroid cell line PCCL3 with FAM83F overexpression and observed that FAM83F deregulates thyroid follicular cell biology leading to loss of thyroid differentiation genes such as Sodium-Iodide Symporter (NIS), reactivation of stem cell markers such as LIN28B and SOX2, induction of cell migration and resistance to doxorubicin-induced apoptosis. Moreover, FAM83F activates MAPK signaling through interaction with BRAF and RAF while impairs TGFß antiproliferative signaling transduction. In this study, we showed FAM83F as a new pro-oncogenic protein overexpressed in thyroid cancer that modulates thyroid follicular cell biology and differentiation through cross-regulation of MAPK and TGFß signaling.

Osteoglycin post-transcriptional regulation by miR-155 induces cellular architecture changes in H9c2 cardiomyoblasts.

Several studies have demonstrated dysregulated cardiac microRNAs (miRNAs) following cardiac stress and development of cardiac hypertrophy and failure. miRNAs are also differentially expressed in the inflammation that occurs in heart failure and, among these inflammatory-related miRNAs, the miR-155 has been implicated in the regulation of cardiac hypertrophy. Despite these data showing the role of miRNA-155 in cardiomyocyte hypertrophy under a hypertrophic stimulus, it is also important to understand the endogenous regulation of this miRNA without a hypertrophic stimulus to fully appreciate its function in this cell type. The first aim of the present study was to determine whether, without a hypertrophic stimulus, miR-155 overexpression induces H9c2 cardiac cells hypertrophy in vitro. The second objective was to determine whether osteoglycin (Ogn), a key regulator of heart mass in rats, mice, and humans, is post-transcriptionally regulated by miR-155 with a potential role in inducing H9c2 cells hypertrophy. Here, we show that, without a hypertrophic stimulus, miR-155 significantly repressed Ogn protein levels, but induce neither alteration in morphological phenotype nor in the expression of the molecular markers that fully characterize pathological hypertrophy of H9c2 cells. However, most importantly, Ogn silencing in H9c2 cells mimicked the effects of miR-155 overexpression in inducing cellular architecture changes that were characterized by a transition of the cell shape from fusiform to rounded. This is a new role of the post-transcriptional regulation of Ogn by miR-155 in the maintenance of the cardiac cell morphology in physiological and pathological conditions.

Osteoglycin inhibition by microRNA miR-155 impairs myogenesis.

Skeletal myogenesis is a regulated process in which mononucleated cells, the myoblasts, undergo proliferation and differentiation. Upon differentiation, the cells align with each other, and subsequently fuse to form terminally differentiated multinucleated myotubes. Previous reports have identified the protein osteoglycin (Ogn) as an important component of the skeletal muscle secretome, which is expressed differentially during muscle development. However, the posttranscriptional regulation of Ogn by microRNAs during myogenesis is unknown. Bioinformatic analysis showed that miR-155 potentially targeted the Ogn transcript at the 3´-untranslated region (3´ UTR). In this study, we tested the hypothesis that miR-155 inhibits the expression of the Ogn to regulate skeletal myogenesis. C2C12 myoblast cells were cultured and miR-155 overexpression or Ogn knockdown was induced by transfection with miR-155 mimic, siRNA-Ogn, and negative controls with lipofectamine for 15 hours. Near confluence (80-90%), myoblasts were induced to differentiate myotubes in a differentiation medium. Luciferase assay was used to confirm the interaction between miR-155 and Ogn 3'UTR. RT-qPCR and Western blot analyses were used to confirm that the differential expression of miR-155 correlates with the differential expression of myogenic molecular markers (Myh2, MyoD, and MyoG) and inhibits Ogn protein and gene expression in myoblasts and myotubes. Myoblast migration and proliferation were assessed using Wound Healing and MTT assays. Our results show that miR-155 interacts with the 3'UTR Ogn region and decrease the levels of Ogn in myotubes. The overexpression of miR-155 increased MyoG expression, decreased myoblasts wound closure rate, and decreased Myh2 expression in myotubes. Moreover, Ogn knockdown reduced the expression levels of MyoD, MyoG, and Myh2 in myotubes. These results reveal a novel pathway in which miR-155 inhibits Ogn expression to regulate proliferation and differentiation of C2C12 myoblast cells.

Identification of Long Noncoding RNAs Deregulated in Papillary Thyroid Cancer and Correlated with BRAFV600E Mutation by Bioinformatics Integrative Analysis.

Papillary Thyroid Cancer (PTC) is an endocrine malignancy in which BRAFV600E oncogenic mutation induces the most aggressive phenotype. In this way, considering that lncRNAs are arising as key players in oncogenesis, it is of high interest the identification of BRAFV600E-associated long noncoding RNAs, which can provide possible candidates for secondary mechanisms of BRAF-induced malignancy in PTC. In this study, we identified differentially expressed lncRNAs correlated with BRAFV600E in PTC and, also, extended the cohort of paired normal and PTC samples to more accurately identify differentially expressed lncRNAs between these conditions. Indirectly validated targets of the differentially expressed lncRNAs in PTC compared to matched normal samples demonstrated an involvement in surface receptors responsible for signal transduction and cell adhesion, as well as, regulation of cell death, proliferation and apoptosis. Targets of BRAFV600E-correlated lncRNAs are mainly involved in calcium signaling pathway, ECM-receptor interaction and MAPK pathway. In summary, our study provides candidate lncRNAs that can be either used for future studies related to diagnosis/prognosis or as targets for PTC management.

MicroRNAs in thyroid development, function and tumorigenesis.

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that modulate the vast majority of cellular processes. During development, the correct timing and expression of miRNAs in the tissue differentiation is essential for organogenesis and functionality. In thyroid gland, DICER and miRNAs are necessary for accurately establishing thyroid follicles and hormone synthesis. Moreover, DICER1 mutations and miRNA deregulation observed in human goiter influence thyroid tumorigenesis. The thyroid malignant transformation by MAPK oncogenes is accompanied by global miRNA changes, with a marked reduction of "tumor-suppressor" miRNAs and activation of oncogenic miRNAs. Loss of thyroid cell differentiation/function, and consequently iodine trapping impairment, is an important clinical characteristic of radioiodine-refractory thyroid cancer. However, few studies have addressed the direct role of miRNAs in thyroid gland physiology. Here, we focus on what we have learned in the thyroid follicular cell differentiation and function as revealed by cell and animal models and miRNA modulation in thyroid tumorigenesis.

MiRNA-146b-5p upregulates migration and invasion of different Papillary Thyroid Carcinoma cells.

BACKGROUND: Tumor invasiveness is directly related to the ability of tumor cells to migrate and invade surrounding tissues, usually degrading extracellular matrix. Despite significant progress in the knowledge about migration and invasion, there is much more to elucidate about their regulatory mechanisms, especially in cancer cells. MicroRNAs (miRs) were recently described as important regulators of migration. Differential expression of miRs in cancer is frequently associated with progression, invasion and metastasis. In papillary thyroid carcinoma (PTC), miR-146b-5p is highly expressed and positively correlated to the degree of malignancy. METHODS: This study aimed to investigate the role of miR-146b-5p on the migratory and invasive behaviors of thyroid cells, using a non tumor rat thyroid follicular cell line (PCCl3) transfected with the miR-146b-5p genomic region, and two PTC cell lines (TPC-1 and BCPAP, bearing distinct oncogenic backgrounds), which express high levels of miR-146b-5p, after miR-146b inhibition by antagomiR and miR-146b overexpression by mimics-miR. Migration and invasion were studied by time-lapse and transwell assays (with and without Matrigel®). Gelatin degradation assays were also employed, as well as F-actin staining. RESULTS: Migration and invasion of PCCl3 were increased 2-3x after miR-146b-5p overexpression (10X) and large lamellipodia were evident in those cells. After miR-146b-5p inhibition, TPC-1 and BCPAP migration and invasion were significantly reduced, with cells showing several simultaneous processes and low polarity. Gelatin degradation was inhibited in TPC-1 cells after inhibition of miR-146b-5p, but was unaffected in BCPAP cells, which did not degrade gelatin. The inhibition of miR-146b-5p in PCCl3 also inhibited migration and invasion, and additional (exogenous) overexpression of this miR in TPC-1 and BCPAP cells increased migration and invasion, without effects on cell morphology or gelatin degradation. The overexpression of SMAD4 in BCPAP cells, a validated target of miR-146b-5p and key protein in the TGF-ß signaling pathway, inhibited migration similarly to the effects observed with the antagomiR 146b-5p. CONCLUSIONS: miR-146b-5p positively regulates migration and invasion of thyroid normal and tumor follicular cells (independently from their original mutation, either BRAF or RET/PTC), through a mechanism that involves the actin cytoskeleton but not an increased capacity of matrix degradation.

MAPK and SHH pathways modulate type 3 deiodinase expression in papillary thyroid carcinoma.

Type 3 deiodinase (DIO3, D3) is reactivated in human neoplasias. Increased D3 levels in papillary thyroid carcinoma (PTC) have been associated with tumor size and metastatic disease. The objective of this study is to investigate the signaling pathways involved in DIO3 upregulation in PTC. Experiments were performed in human PTC cell lines (K1 and TPC-1 cells) or tumor samples. DIO3 mRNA and activity were evaluated by real-time PCR and ion-exchange column chromatography respectively. Western blot analysis was used to determine the levels of D3 protein. DIO3 gene silencing was performed via siRNA transfection. DIO3 mRNA levels and activity were readily detected in K1 (BRAF(V6) (0) (0E)) and, at lower levels, in TPC-1 (RET/PTC1) cells (P<0.007 and P=0.02 respectively). Similarly, DIO3 mRNA levels were higher in PTC samples harboring the BRAF(V600E) mutation as compared with those with RET/PTC1 rearrangement or negative for these mutations (P<0.001). Specific inhibition of BRAF oncogene (PLX4032, 3 µM), MEK (U0126, 10-20 µM) or p38 (SB203580, 10-20 µM) signaling was associated with decreases in DIO3 expression in K1 and TPC-1 cells. Additionally, the blockage of the sonic hedgehog (SHH) pathway by cyclopamine (10  µM) resulted in markedly decreases in DIO3 mRNA levels. Interestingly, siRNA-mediated DIO3 silencing induced decreases on cyclin D1 expression and partial G1 phase cell cycle arrest, thereby downregulating cell proliferation. In conclusion, sustained activation of the MAPK and SHH pathways modulate the levels of DIO3 expression in PTC. Importantly, DIO3 silencing was associated with decreases in cell proliferation, thus suggesting a D3 role in tumor growth and aggressiveness.