PCCL3 Cells Research Articles

Differential regulation of monocarboxylate transporter 8 expression in thyroid cancer and hyperthyroidism.

Thyroid hormone (TH) transporters are expressed in thyrocytes and most play a role in TH release. We asked whether expression of the monocarboxylate transporter 8 (MCT8) and the L-type amino acid transporters LAT2 and LAT4 is changed with thyrocyte dedifferentiation and in hyperfunctioning thyroid tissues. Protein expression and localization of transporters was determined by immunohistochemistry in human thyroid specimen including normal thyroid tissue (NT, n = 19), follicular adenoma (FA, n = 44), follicular thyroid carcinoma (FTC, n = 45), papillary thyroid carcinoma (PTC, n = 40), anaplastic thyroid carcinoma (ATC, n = 40) and Graves' disease (GD, n = 50) by calculating the 'hybrid' (H) score. Regulation of transporter expression was investigated in the rat follicular thyroid cell line PCCL3 under basal and thyroid stimulating hormone (TSH) conditions. MCT8 and LAT4 were localized at the plasma membrane, while LAT2 transporter showed cytoplasmic localization. MCT8 expression was downregulated in benign and malignant thyroid tumours as compared to NT. In contrast, significant upregulation of MCT8, LAT2 and LAT4 was found in GD. Furthermore, a stronger expression of MCT8 was demonstrated in PCCL3 cells after TSH stimulation. Downregulation of MCT8 in thyroid cancers qualifies MCT8 as a marker of thyroid differentiation. The more variable expression of LATs in distinct thyroid malignancies may be linked with other transporter properties relevant to altered metabolism in cancer cells, i.e. amino acid transport. Consistent upregulation of MCT8 in GD is in line with increased TH release in hyperthyroidism, an assumption supported by our in vitro results showing TSH-dependent upregulation of MCT8.

Signal Transducer and Activator of Transcription 1 Plays a Pivotal Role in RET/PTC3 Oncogene-induced Expression of Indoleamine 2,3-Dioxygenase 1

Indoleamine 2,3-dioxygenase 1 (IDO1) is a single chain oxidoreductase that catalyzes tryptophan degradation to kynurenine. In cancer, it exerts an immunosuppressive function as part of an acquired mechanism of immune escape. Recently, we demonstrated that IDO1 expression is significantly higher in all thyroid cancer histotypes compared with normal thyroid and that its expression levels correlate with T regulatory (Treg) lymphocyte densities in the tumor microenvironment. BRAFV600E- and RET/PTC3-expressing PcCL3 cells were used as cellular models for the evaluation of IDO1 expression in thyroid carcinoma cells and for the study of involved signal transduction pathways. BRAFV600E-expressing PcCL3 cells did not show IDO1 expression. Conversely, RET/PTC3-expressing cells were characterized by a high IDO1 expression. Moreover, we found that, the STAT1-IRF1 pathway was instrumental for IDO1 expression in RET/PTC3 expressing cells. In detail, RET/PTC3 induced STAT1 overexpression and phosphorylation at Ser-727 and Tyr-701. STAT1 transcriptional regulation appeared to require activation of the canonical NF-κB pathway. Conversely, activation of the MAPK and PI3K-AKT pathways primarily regulated Ser-727 phosphorylation, whereas a physical interaction between RET/PTC3 and STAT1, followed by a direct tyrosine phosphorylation event, was necessary for STAT1 Tyr-701 phosphorylation. These data provide the first evidence of a direct link between IDO1 expression and the oncogenic activation of RET in thyroid carcinoma and describe the involved signal transduction pathways. Moreover, they suggest possible novel molecular targets for the abrogation of tumor microenvironment immunosuppression. The detection of those targets is becoming increasingly important to yield the full function of novel immune checkpoint inhibitors.

Bromodomain and Extraterminal Protein Inhibitor JQ1 Suppresses Thyroid Tumor Growth in a Mouse Model.

New therapeutic approaches are needed for patients with thyroid cancer refractory to radioiodine treatment. An inhibitor of bromodomain and extraterminal domain (BET) proteins, JQ1, shows potent antitumor effects in hematological cancers and solid tumors. To evaluate whether JQ1 is effective against thyroid cancer, we examined antitumor efficacy of JQ1 using the ThrbPV/PVKrasG12D mouse, a model of anaplastic thyroid cancer. We treated ThrbPV/PVKrasG12D mice with vehicle or JQ1 at a dose of 50 mg/kg body weight/day starting at the age of 8 weeks for a 10-week period and monitored thyroid tumor progression. JQ1 markedly inhibited thyroid tumor growth and prolonged survival of these mice. Global differential gene expression analysis showed that JQ1 suppressed the cMyc (hereafter referred to as Myc) transcription program by inhibiting mRNA expression of Myc, ccnd1, and other related genes. JQ1-suppressed Myc expression was accompanied by chromatin remodeling as evidenced by increased expression of histones and hexamethylene bis-acetamide inducible 1, a suppressor of RNA polymerase II transcription elongation. Analyses showed that JQ1 decreased MYC abundance in thyroid tumors and attenuated the cyclin D1-CDK4-Rb-E2F3 signaling to decrease tumor growth. Further analysis indicated that JQ1 inhibited the recruitment of BDR4 to the promoter complex of the Myc and Ccnd1 genes in rat thyroid follicular PCCL3 cells, resulting in decreased MYC expression at the mRNA and protein levels to inhibit tumor cell proliferation. These preclinical findings suggest that BET inhibitors may be an effective agent to reduce thyroid tumor burden for the treatment of refractory thyroid cancer. Clin Cancer Res; 23(2); 430-40. ©2016 AACR.

Comparison of clonogenic cell survival and DNA damage induced by 188Re and X-rays in rat thyroid cells.

Ionizing radiation produces DNA lesions among which DNA double strand breaks (DSB) are the most critical events. Radiation of various energy types might differ in their biological effectiveness. Here, we compared cell survival and DNA damage induced by 188Re and X-rays using γH2AX foci as a measure of DSB. The correlation between survival and residual foci was also analyzed. PCCl3 cells were irradiated with 200 kV X-rays (1.2 Gy/min) or 0.5-25 MBq/ml 188Re (1 h irradiation) achieving doses up to 10 Gy. By blocking of sodium iodide symporter (NIS) essentially extracellular activity could be guaranteed. Survival fractions (SF) were detected by colony forming assay. Initial and residual γH2AX foci (15 min and 24 h after irradiation) were assessed by immunostaining. The relationship between SF and residual radiation induced γH2AX foci (RIF) was evaluated by Spearman and Pearson correlation tests. We did not find significant differences between the survival curves in terms of the radiation quality. The D37 values were 4.6 Gy and 4.2 Gy for 188Re or X-ray, respectively. The initial foci numbers were in the same range for 188Re and X-ray, but higher levels of residual foci persisted after X-rays in comparison to 188Re (1 GyX-ray 6.5 ± 0.2; 1 GyRe-188 4.8 ± 0.2 RIF). Accordingly, for 188Re a higher extent of DSB repair was found. The Spearman test revealed a significant (p < 0.01) correlation between SF and residual RIF for both radiation modalities. No differences in terms of radiation were found for SF and initial foci. However, residual foci were lower for 188Re than for X-rays. A prediction of SF by residual foci should consider the properties of the radiation qualities that influence foci removal and DSB repair.

3-Iodothyronamine Decreases Expression of Genes Involved in Iodide Metabolism in Mouse Thyroids and Inhibits Iodide Uptake in PCCL3 Thyrocytes.

3-Iodothyronamine (3-T1AM) is an endogenous decarboxylated thyroid hormone (TH) metabolite. Pharmacological doses of 3-T1AM decrease heart rate, body temperature, and metabolic rate in rodents-effects that are contrary to classic TH excess. Furthermore, a single dose of 3-T1AM was shown to suppress the hypothalamic-pituitary-thyroid (HPT) axis in rats. It was hypothesized that 3-T1AM might play a role in the fine-tuning of TH action and might have a direct regulatory effect on the thyroid gland. This study tested whether repeated 3-T1AM treatment interfered with thyroid function and the HPT axis in mice. Therefore, male C57BL/6 mice were intraperitoneally injected with 5 mg/kg of 3-T1AM or vehicle daily for seven days. Additionally, the effects of 3-T1AM on the differentiated rat thyrocyte cell line PCCL3 were analyzed. Repeated administration of 3-T1AM decreased thyroidal mRNA content of the sodium iodide symporter (Nis), thyroglobulin, and pendrin in mice. No interference with the HPT axis was observed, as determined by unaltered pituitary mRNA levels of triiodothyronine-responsive genes, including thyrotropin subunit β. Furthermore, 3-T1AM treatment did not change transcript levels of hepatic triiodothyronine-responsive genes, such as deiodinase 1. In line with this, serum TH concentrations were not changed after the treatment period of seven days. In concordance with the in vivo findings, 3-T1AM decreased the thyrotropin-dependent expression of Nis and functional iodide uptake in PCCL3 cells in vitro. Additionally, uptake and metabolism of 3-T1AM by PCCL3 cells was observed, as well as 3-T1AM-dependent changes in intracellular Ca2+ concentration that might be involved in mediating the reported effects. In conclusion, 3-T1AM application decreased expression of selected TH synthesis genes by acting directly on the thyroid gland, and it might therefore affect TH synthesis without involvement of the HPT axis.

Genomic binding of PAX8-PPARG fusion protein regulates cancer-related pathways and alters the immune landscape of thyroid cancer.

PAX8-PPARG fusion protein (PPFP) results from a t(2;3)(q13;p25) chromosomal translocation, is found in 30% of follicular thyroid carcinomas, and demonstrates oncogenic capacity in transgenic mice. A PPARG ligand, pioglitazone, is highly therapeutic in mice with PPFP thyroid cancer. However, only limited data exist to characterize the binding sites and oncogenic function of PPFP, or to explain the observed therapeutic effect of pioglitazone. Here we used our previously characterized transgenic mouse model of PPFP follicular thyroid carcinoma to identify PPFP binding sites in vivo using ChIP-seq, and to distinguish genes and pathways regulated directly or indirectly by PPFP with and without pioglitazone treatment via integration with RNA-seq data. PPFP bound to DNA regions containing the PAX8 and/or the PPARG motif, near genes involved in lipid metabolism, the cell cycle, apoptosis, and cell motility; the binding site distribution was highly concordant with our previous study in a rat PCCL3 cell line. Most strikingly, pioglitazone induced an immune cell infiltration including macrophages and T cells only in the presence of PPFP, which may be central to its therapeutic effect.

Loss of tyrosine phosphorylation at Y406 abrogates the tumor suppressor functions of the thyroid hormone receptor β.

We have recently identified that phosphorylation at tyrosine (Y)406 is critical for the tumor suppressor functions of the thyroid hormone receptor β1 (TRβ) in a breast cancer line. However, still unclear is whether the critical tumor suppressor role of phosphorylated Y406 of TRβ is limited to only breast cancer cells or could be extended to other cell types. In the present studies, we addressed this question by stably expressing TRβ, a mutated TRβ oncogene (PV), or a TRβ mutated at Y406 (TRβY406F) in rat PCCL3 thyroid follicular cells and evaluated their tumor characteristics in athymic mice with elevated thyroid stimulating hormone. PCCL3 cells stably expressing PV (PCCL3-PV), TRβY406F (PCCL3-TRβY406F), or vector only (PCCL3-Neo) developed tumors with sizes in the rank order of TRβY406F>PV = Neo, whereas PCCL3 cells expressing TRβ (PCCL3-TRβ) barely developed tumors. As evidenced by markedly elevated Ki67, cyclin D1, and p-Rb protein abundance, proliferative activity was high in PV and TRβY406F tumors, but low in TRβ tumors. These results indicate that TRβ acted as a tumor suppressor in PCCL3 cells, whereas TRβY406F and PV had lost tumor suppressor activity. Interestingly, TRβY406F tumors had very low necrotic areas with decreased TNFα-NFκB signaling to lower apoptotic activity. In contrast, PV tumors had prominent large necrotic areas, with no apparent changes in TNFα-NFκB signaling, indicating distinct oncogenic activities of mutant PV and TRβY406F. Thus, the present studies uncovered a novel mechanism by which TRβ could function as a tumor suppressor through modulation of the TNFα-NFκB signaling. © 2016 Wiley Periodicals, Inc.

The effect of dimethyl sulfoxide on the induction of DNA strand breaks in plasmid DNA and colony formation of PC Cl3 mammalian cells by alpha-, beta-, and Auger electron emitters (223)Ra, (188)Re, and (99m)Tc.

BackgroundDNA damage occurs as a consequence of both direct and indirect effects of ionizing radiation. The severity of DNA damage depends on the physical characteristics of the radiation quality, e.g., the linear energy transfer (LET). There are still contrary findings regarding direct or indirect interactions of high-LET emitters with DNA. Our aim is to determine DNA damage and the effect on cellular survival induced by 223Ra compared to 188Re and 99mTc modulated by the radical scavenger dimethyl sulfoxide (DMSO).MethodsRadioactive solutions of 223Ra, 188Re, or 99mTc were added to either plasmid DNA or to PC Cl3 cells in the absence or presence of DMSO. Following irradiation, single strand breaks (SSB) and double strand breaks (DSB) in plasmid DNA were analyzed by gel electrophoresis. To determine the radiosensitivity of the rat thyroid cell line (PC Cl3), survival curves were performed using the colony formation assay.ResultsExposure to 120 Gy of 223Ra, 188Re, or 99mTc leads to maximal yields of SSB (80 %) in plasmid DNA. Irradiation with 540 Gy 223Ra and 500 Gy 188Re or 99mTc induced 40, 28, and 64 % linear plasmid conformations, respectively. DMSO prevented the SSB and DSB in a similar way for all radionuclides. However, with the α-emitter 223Ra, a low level of DSB could not be prevented by DMSO. Irradiation of PC Cl3 cells with 223Ra, 188Re, and 99mTc pre-incubated with DMSO revealed enhanced survival fractions (SF) in comparison to treatment without DMSO. Protection factors (PF) were calculated using the fitted survival curves. These factors are 1.23 ± 0.04, 1.20 ± 0.19, and 1.34 ± 0.05 for 223Ra, 188Re, and 99mTc, respectively.ConclusionsFor 223Ra, as well as for 188Re and 99mTc, dose-dependent radiation effects were found applicable for plasmid DNA and PC Cl3 cells. The radioprotection by DMSO was in the same range for high- and low-LET emitter. Overall, the results indicate the contribution of mainly indirect radiation effects for each of the radionuclides regarding DNA damage and cell survival. In summary, our findings may contribute to fundamental knowledge about the α-particle induced DNA damage.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-016-0203-x) contains supplementary material, which is available to authorized users.

Excess iodide downregulates Na+/I− symporter gene transcription through activation of PI3K/Akt pathway

Transcriptional mechanisms associated with iodide-induced downregulation of NIS expression remain uncertain. Here, we further analyzed the transcriptional regulation of NIS gene expression by excess iodide using PCCl3 cells. NIS promoter activity was reduced in cells treated for 12–24 h with 10−5 to 10−3 M NaI. Site-directed mutagenesis of Pax8 and NF-κB cis-acting elements abrogated the iodide-induced NIS transcription repression. Indeed, excess iodide (10−3 M) excluded Pax8 from the nucleus, decreased p65 total expression and reduced their transcriptional activity. Importantly, p65-Pax8 physical interaction and binding to NIS upstream enhancer were reduced upon iodide treatment. PI3K/Akt pathway activation by iodide-induced ROS production is involved in the transcriptional repression of NIS expression. In conclusion, the results indicated that excess iodide transcriptionally represses NIS gene expression through the impairment of Pax8 and p65 transcriptional activity. Furthermore, the data presented herein described novel roles for PI3K/Akt signaling pathway and oxidative status in the thyroid autoregulatory phenomenon.

Iodide excess regulates its own efflux: a possible involvement of pendrin.

Adequate iodide supply and metabolism are essential for thyroid hormones synthesis. In thyrocytes, iodide uptake is mediated by the sodium-iodide symporter, but several proteins appear to be involved in iodide efflux. Previous studies demonstrated that pendrin is able to mediate apical efflux of iodide in thyrocytes. Acute iodide excess transiently impairs thyroid hormone synthesis, a phenomenon known as the Wolff-Chaikoff effect. Although the escape from this inhibitory effect is not completely understood, it has been related to the inhibition of sodium-iodide symporter-mediated iodide uptake. However, the effects of iodide excess on iodide efflux have not been characterized. Herein, we investigated the consequences of iodide excess on pendrin abundance, subcellular localization, and iodide efflux in rat thyroid PCCl3 cells. Our results indicate that iodide excess increases pendrin abundance and plasma membrane insertion after 24 h of treatment. Moreover, iodide excess increases pendrin half-life. Finally, iodide exposure also increases iodide efflux from PCCl3 cells. In conclusion, these data suggest that pendrin may have an important role in mediating iodide efflux in thyrocytes, especially under conditions of iodide excess.

Clinical relevance of thyroid cell models in redox research.

BackgroundThyroid-derived cell models are commonly used to investigate the characteristics of thyroid cancers. It is noteworthy that each in vitro single cell model system imitates only a few characteristics of thyroid cancer depending on e.g. source of cells or oncogene used to transform the cells.MethodsIn the current work we utilized rat thyroid cancer cell models to determine their clinical relevance in redox gene studies by comparing in vitro expression data to thyroid Oncomine microarray database. To survey the cell lines we analyzed mRNA expression of genes that produce superoxide anion (nox family), genes that catalyze destruction of superoxide anion to hydrogen peroxide (sod family), and genes that remove hydrogen peroxide from cellular environment (catalase, gpx family and prdx family).ResultsBased on the current results, rat thyroid PC Cl3, PC PTC1, PC E1A, or FRLT5 cell models can be used to study NOX2, NOX4, SOD2, SOD3, CATALASE, GPX1, GPX2, GPX5, PRDX2, and PRDX3 gene expression and function.ConclusionsRedox gene expression in rat originated single cell model systems used to study human thyroid carcinogenesis corresponds only partly with human redox gene expression, which may be caused by differences in redox gene activation stimulus. The data suggest careful estimation of the data observed in rat thyroid in vitro models.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-015-0264-3) contains supplementary material, which is available to authorized users.

The Coiled-Coil Domain Containing 80 (ccdc80) gene regulates gadd45β2 expression in the developing somites of zebrafish as a new player of the hedgehog pathway.

The Coiled-Coil Domain Containing 80 (CCDC80) gene has been identified as strongly induced in rat thyroid PC CL3 cells immortalized by the adenoviral E1A gene. In human, CCDC80 is a potential oncosoppressor due to its down-regulation in several tumor cell lines and tissues and it is expressed in almost all tissues. CCDC80 has homologous in mouse, chicken, and zebrafish. We cloned the zebrafish ccdc80 and analyzed its expression and function during embryonic development. The in-silico translated zebrafish protein shares high similarity with its mammalian homologous, with nuclear localization signals and a signal peptide. Gene expression analysis demonstrates that zebrafish ccdc80 is maternally and zygotically expressed throughout the development. In particular, ccdc80 is strongly expressed in the notochord and it is under the regulation of the Hedgehog pathway. In this work we investigated the functional effects of ccdc80-loss-of-function during embryonic development and verified its interaction with gadd45β2 in somitogenesis.

MicroRNA-339-5p modulates Na+/I- symporter-mediated radioiodide uptake.

Na(+)/I(-) symporter (NIS)-mediated radioiodide uptake (RAIU) serves as the basis for targeted ablation of thyroid cancer remnants. However, many patients with thyroid cancer have reduced NIS expression/function and hence do not benefit from radioiodine therapy. microRNA (miR) has emerged as a promising therapeutic target in many diseases; yet, the role of miRs in NIS-mediated RAIU has not been investigated. In silico analysis was used to identify miRs that may bind to the 3'UTR of human NIS (hNIS). The top candidate miR-339-5p directly bound to the 3'UTR of hNIS. miR-339-5p overexpression decreased NIS-mediated RAIU in HEK293 cells expressing exogenous hNIS, decreased the levels of NIS mRNA, and RAIU in transretinoic acid/hydrocortisone (tRA/H)-treated MCF-7 human breast cancer cells as well as thyrotropin-stimulated PCCl3 rat thyroid cells. Nanostring nCounter rat miR expression assay was conducted to identify miRs deregulated by TGFβ, Akti-1/2, or 17-AAG known to modulate RAIU in PCCl3 cells. Among 38 miRs identified, 18 were conserved in humans. One of the 18 miRs, miR-195, was predicted to bind to the 3'UTR of hNIS and its overexpression decreased RAIU in tRA/H-treated MCF-7 cells. miR-339-5p was modestly increased in most papillary thyroid carcinomas (PTCs), yet miR-195 was significantly decreased in PTCs. Interestingly, the expression profiles of 18 miRs could be used to distinguish most PTCs from nonmalignant thyroid tissues. This is the first report, to our knowledge, demonstrating that hNIS-mediated RAIU can be modulated by miRs, and that the same miRs may also play roles in the development or maintenance of thyroid malignancy. Accordingly, miRs may serve as emerging targets to halt the progression of thyroid cancer and to enhance the efficacy of radioiodine therapy.

Anoctamin-1/TMEM16A is the major apical iodide channel of the thyrocyte.

Iodide is captured by thyrocytes through the Na(+)/I(-) symporter (NIS) before being released into the follicular lumen, where it is oxidized and incorporated into thyroglobulin for the production of thyroid hormones. Several reports point to pendrin as a candidate protein for iodide export from thyroid cells into the follicular lumen. Here, we show that a recently discovered Ca(2+)-activated anion channel, TMEM16A or anoctamin-1 (ANO1), also exports iodide from rat thyroid cell lines and from HEK 293T cells expressing human NIS and ANO1. The Ano1 mRNA is expressed in PCCl3 and FRTL-5 rat thyroid cell lines, and this expression is stimulated by thyrotropin (TSH) in rat in vivo, leading to the accumulation of the ANO1 protein at the apical membrane of thyroid follicles. Moreover, ANO1 properties, i.e., activation by intracellular calcium (i.e., by ionomycin or by ATP), low but positive affinity for pertechnetate, and nonrequirement for chloride, better fit with the iodide release characteristics of PCCl3 and FRTL-5 rat thyroid cell lines than the dissimilar properties of pendrin. Most importantly, iodide release by PCCl3 and FRTL-5 cells is efficiently blocked by T16Ainh-A01, an ANO1-specific inhibitor, and upon ANO1 knockdown by RNA interference. Finally, we show that the T16Ainh-A01 inhibitor efficiently blocks ATP-induced iodide efflux from in vitro-cultured human thyrocytes. In conclusion, our data strongly suggest that ANO1 is responsible for most of the iodide efflux across the apical membrane of thyroid cells.

Abstract 5210: MicroRNA miR-17-92 impairs TGFβ signaling responsiveness in BRAF oncogene-activated thyroid cells

Abstract Background: Thyroid follicular cells proliferation is regulated by TGFβ anti-mitogenic signal. However, thyroid cancer cells become refractory to this inhibitory signal. TGFβ signaling deregulation is observed in thyroid cancer, which shows high frequence of MAPK genetic alteration. BRAF mutation (BRAFT1799A), the most prevalent alteration in thyroid cancer, induces MAPK activtion and thyroid oncogenesis. In this process, deregulation of microRNAs potentiates oncogene activation. MicroRNAs are small non-coding RNAs that regulate protein levels by pairing to 3-UTR of mRNA and blocking translation. miR-17-92 cluster of microRNAs, miR-17-5p/3p, miR-18a, miR-19a/b, miR20a and miR-92a, regulates several tumor-suppressor genes and is predicted to target TGFβ pathway components. Therefore, we aim at analyzing the influence of miR-17-92 in TGFβ signaling during oncogene activation in thyroid follicular cells and its implication for thyroid cancer biology. Methods: The BRAF9-6 cells, derived from normal rat thyroid follicular cell PCCl3, express BRAFT1799A under doxicyclin (dox) stimulation. The PCCl3-miR-17-92 cells, derived from PCCl3 cells, over-express miR-17-92 cluster (pCDNA3.1-Neo). PCCl3- is the control cell transfected with empty plasmid. MiR-17-92 expression was detected by real-time qPCR. Cell viability was measured using MTT assay and proliferation by cell counting (24-72h). TGFβ signaling components, Smad4 and Tgfbr2, protein levels was analyzed by Western-blotting. TGFβ responsiveness assay was analyzed by luciferase gene reporter assay after transfection of 3TP-lux plasmid and treatment with 1-2ng/mL recombinant TGFβ for 24 hours. Results: BRAF-oncogene activation (dox) in thyroid follicular cells induced a high expression of miR-17-92 cluster components and reduced Smad4 and Tgfbr2 protein levels. In order to analyze the particular influence of miR-17-92 in TGFβ signaling, we constructed a cell line, PCCl3-miR-17-92, that over-expressed miR-17-92 in normal thyroid follicular cells. Over-expression of miR-17-92 inhibited Tgfbr2 and Smad4 protein levels that reflected in impaired responsiveness to recombinant TGFβ inhibitory signal. Using the 3TP-lux reporter gene assay, that contains TGFβ signaling activation responsive sequences, we observed that recombinant TGFβ treatment in PCCl3- cells resulted in a 8.0 and 12.3-fold increase in luciferase activity at concentrations of 1ng/ml and 2ng/mL of recombinant TGFβ, respectively, while a 4.3 and 5.6-fold increase is observed in PCCl3-miR-17-92 cells. Moreover, PCCl3-miR-17-92 showed increases cell proliferation and cell viability in 18% and 9%, respectively, in comparison to PCCL3-. Conclusion: miR-17-92 over-expression impairs normal thyroid follicular cells responsiveness to TGFβ and increases cell proliferation and viability, which could imply in TGFβ inhibitory signal refractoriness observed in thyroid cancer cells. Citation Format: Cesar Seigi Fuziwara, Felipe E. Martins, Edna T. Kimura. MicroRNA miR-17-92 impairs TGFβ signaling responsiveness in BRAF oncogene-activated thyroid cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5210. doi:10.1158/1538-7445.AM2014-5210

Synergistic Signaling of KRAS and Thyroid Hormone Receptor β Mutants Promotes Undifferentiated Thyroid Cancer through MYC Up-Regulation

Undifferentiated thyroid carcinoma is one of the most aggressive human cancers with frequent RAS mutations. How mutations of the RAS gene contribute to undifferentiated thyroid cancer remains largely unknown. Mice harboring a potent dominant negative mutant thyroid hormone receptor β, TRβPV (ThrbPV/PV), spontaneously develop well-differentiated follicular thyroid cancer similar to human cancer. We genetically targeted the KrasG12D mutation to thyroid epithelial cells of ThrbPV/PV mice to understand how KrasG12D mutation could induce undifferentiated thyroid cancer in ThrbPV/PVKrasG12D mice. ThrbPV/PVKrasG12D mice exhibited poorer survival due to more aggressive thyroid tumors with capsular invasion, vascular invasion, and distant metastases to the lung occurring at an earlier age and at a higher frequency than ThrbPV/PV mice did. Importantly, ThrbPV/PVKrasG12D mice developed frequent anaplastic foci with complete loss of normal thyroid follicular morphology. Within the anaplastic foci, the thyroid-specific transcription factor paired box gene 8 (PAX8) expression was virtually lost and the loss of PAX8 expression was inversely correlated with elevated MYC expression. Consistently, co-expression of KRASG12D with TRβPV upregulated MYC levels in rat thyroid pccl3 cells, and MYC acted to enhance the TRβPV-mediated repression of the Pax8 promoter activity of a distant upstream enhancer, critical for thyroid-specific Pax8 expression. Our findings indicated that synergistic signaling of KRASG12D and TRβPV led to increased MYC expression. Upregulated MYC contributes to the initiation of undifferentiated thyroid cancer, in part, through enhancing TRβPV-mediated repression of the Pax8 expression. Thus, MYC might serve as a potential target for therapeutic intervention.

In vitro screening of thyroid function disruption: PC-Cl3 cell line, a promising tool

In vitro screening of thyroid function disruption: PC-Cl3 cell line, a promising tool

Switch in signaling control of mTORC1 activity after oncoprotein expression in thyroid cancer cell lines.

Thyroid growth is regulated by TSH and requires mammalian target of rapamycin (mTOR). Thyroid cancers frequently exhibit mutations in MAPK and/or phosphoinositol-3-kinase-related kinase effectors. The objective of the study was to explore the contribution of RET/PTC, RAS, and BRAF to mTOR regulation and response to mTOR inhibitors. PCCL3 cells conditionally expressing RET/PTC3, HRAS(G12V), or BRAF(V600E) and human thyroid cancer cells harboring mutations of these genes were used to test pathways controlling mTOR and its requirement for growth. TSH/cAMP-induced growth of PCCL3 cells requires mTOR, which is stimulated via protein kinase A in a MAPK kinase (MEK)- and AKT-independent manner. Expression of RET/PTC3, HRAS(G12V), or BRAF(V600E) in PCCL3 cells induces mTOR but does not entirely abrogate the cAMP-mediated control of its activity. Acute oncoprotein-induced mTOR activity is regulated by MEK and AKT, albeit to differing degrees. By contrast, mTOR was not activated by TSH/cAMP in human thyroid cancer cells. Tumor genotype did not predict the effects of rapamycin or the mTOR kinase inhibitor AZD8055 on growth, with the exception of a PTEN-null cell line. Selective blockade of MEK did not influence mTOR activity of BRAF or RAS mutant cells. Combined MEK and mTOR kinase inhibition was synergistic on growth of BRAF- and RAS-mutant thyroid cancer cells in vitro and in vivo. Thyroid cancer cells lose TSH/cAMP dependency of mTOR signaling and cell growth. mTOR activity is not decreased by the MEK or AKT inhibitors in the RAS or BRAF human thyroid cancer cell lines. This may account for the augmented effects of combining the mTOR inhibitors with selective antagonists of these oncogenic drivers.

AMP-Activated Protein Kinase Activation Leads to Lysome-Mediated NA+/Iˉ-Symporter Protein Degradation in Rat Thyroid Cells

Iodide uptake by thyroid cells is mediated by a transmembrane glycoprotein known as the Na+/I--symporter (NIS). NIS-mediated iodide uptake plays important physiological role in thyroid gland function, as well as in diagnostic and treatment of Graves' disease and thyroid cancer. Although different studies investigated the transcriptional mechanisms of NIS expression, there is no report on the NIS post-translational regulation related to NIS protein degradation in thyroid cells. Recently, our group showed that AMP-activated protein kinase (AMPK) plays a pivotal role in the rat thyroid gland, downregulating iodide uptake, NIS protein, and mRNA content. Since several studies demonstrated that AMPK regulates post-transcriptional mechanisms, such as autophagy-mediated processes in different tissues, we hypothesized that AMPK activation could also regulate NIS protein degradation through the lysosome pathway in thyroid cells. Rat follicular thyroid PCCL3 cells cultivated in Ham's F12 supplemented with 5% calf serum and hormones were exposed to the AMPK pharmacological activator 5-aminoimidazole-4 carboxamide ribonucleoside (AICAR), in the presence or absence of Bafilomycin A1 or MG132 for 24 h. Treatment of PCCL3 cells with Bafilomycin A1 fully prevented the decrease of iodide uptake and NIS protein content mediated by AMPK activation. In contrast, the treatment with MG132 was unable to prevent the effects of AMPK activation on NIS. Our results show that AMPK activation significantly induces NIS protein degradation through a lysosome-mediated mechanism.

A New Mutation in the Promoter Region of the PAX8 Gene Causes True Congenital Hypothyroidism with Thyroid Hypoplasia in a Girl with Down's Syndrome

Thyroid dysfunction is common in newborn infants with Down's syndrome (DS), but defects causing classic thyroid dysgenesis (TD) with permanent congenital hypothyroidism (CH) have not been described. We studied a girl with DS and CH who had a mutation in the promoter sequence of the PAX8 gene. A female infant was found to have trisomy 21 and CH, with a venous thyrotropin (TSH) of >150 mU/L and a free thyroxine (fT4) of 15.1 pmol/L (day 12). Thyroid peroxidase antibodies and thyroglobulin antibodies were elevated. Scintigraphy showed normal uptake, but ultrasound identified a small gland with heterogenous echotexture and cystic changes. Sequence analysis of the PAX8 gene revealed a new heterozygous maternally inherited mutation (-3C>T) close to the transcription initiation site. Electromobility shift assay studies of the wild type and the mutant PAX8 sequence incubated with nuclear extracts from PCCL3 cells exhibited that the sequence at position -3 is not involved in specific protein binding. However, the mutant PAX8 promoter showed a significantly reduced transcriptional activation of a luciferase reporter gene in vitro tested in HEK, PCCL3, as well as in HeLa cells, indicating that this mutation is very likely to lead to reduced PAX8 expression. The persistent CH in this patient with DS is likely to be attributable to the diminished PAX8 expression due to a new heterozygous mutation in the PAX8 promoter sequence. Our case shows that true CH may occur in DS, as in the general population. Furthermore, it is possible that the trisomy 21 itself may have resulted in a more severe phenotypic expression of the PAX8 mutation in the child than the mother.