Compelling epidemiological evidence shows a strong positive correlation of obesity with thyroid cancer. In vivo studies have provided molecular evidence that high-fat-diet-induced obesity promotes thyroid cancer progression by aberrantly activating leptin-JAK2-STAT3 signaling in a mouse model of thyroid cancer (Thrb PV/PV Pten +/ − mice). The Thrb PV/PV Pten +/ − mouse expresses a dominantly negative thyroid hormone receptor β (denoted as PV) and a deletion of one single allele of the Pten gene. The Thrb PV/PV Pten +/ − mouse spontaneously develops follicular thyroid cancer, which allows its use as a preclinical mouse model to test potential therapeutics. We recently showed that inhibition of STAT3 activity by a specific inhibitor markedly delays thyroid cancer progression in high-fat-diet-induced obese Thrb PV/PV Pten +/ − mice (HFD-Thrb PV/PV Pten +/ − mice). Further, metformin, a widely used antidiabetic drug, blocks invasion and metastasis, but not thyroid tumor growth in HFD-Thrb PV/PV Pten +/ − mice. To improve efficacy in reducing thyroid tumor growth, we treated HFD-Thrb PV/PV Pten +/ − with JQ1, a potent inhibitor of the activity of bromodomain and extraterminal domain (BET) and with metformin. We found that the combined treatment synergistically suppressed thyroid tumor growth by attenuating STAT3 and ERK signaling, resulting in decreased anti-apoptotic key regulators such as Mcl-1, Bcl-2 and survivin and increased pro-apoptotic regulators such as Bim, BAD and cleave caspase 3. Furthermore, combined treatment of JQ1 and metformin reduced cMyc protein levels to suppress vascular invasion, anaplasia and lung metastasis. These findings indicate that combined treatment is more effective than metformin alone and suggest a novel treatment modality for obesity-activated thyroid cancer.
Sunmi Park, Mark C Willingham, Jun Qi, and Sheue-Yann Cheng
Iñigo Landa, Cristina Montero-Conde, Donatella Malanga, Silvia De Gisi, Guillermo Pita, Luis-Javier Leandro-García, Lucía Inglada-Pérez, Rocío Letón, Carmela De Marco, Cristina Rodríguez-Antona, Giuseppe Viglietto, and Mercedes Robledo
The aim of this study is to assess if common genetic variants located in the CDKN1B locus, coding for the cell cycle inhibitor p27Kip1, are involved in thyroid cancer susceptibility. Based on the literature and functional predictions, we selected three polymorphisms within the CDKN1B gene (rs2066827 (T326G, V109G), rs34330 (−79C>T) and rs36228499 (−838C>A)) to perform the first case–control study in thyroid cancer involving this locus. We had 649 Spanish patients with sporadic thyroid cancer and 385 healthy representative controls available. Luciferase reporter gene assays, real-time quantitative reverse transcription-PCR and immunoblot experiments were carried out to demonstrate the putative effect of the associated variant. The polymorphism rs34330 (−79C>T) was identified as a risk factor for developing the follicular variant of papillary thyroid carcinoma (FVPTC), fitting a recessive model (odds ratio=2.12; 95% confidence interval=1.09–4.15; P value=0.023). The risk allele (T) of this single nucleotide polymorphism led to a lower transcription rate in cells transfected with a luciferase reporter driven by the polymorphic p27Kip1 promoter (P value <0.001). This effect was observed in −79TT genotype control carriers, who showed a tendency towards lower CDKN1B mRNA levels in lymphocytes, as well as at the protein level. This is the first study that identifies CDKN1B as a low-penetrance gene in thyroid cancer, and specifically in FVPTC subtype. We propose a reduced CDKN1B gene transcription depending on the genotype of the −79C>T (rs34330) variant as a novel mechanism underlying p27Kip1 downregulation.
Kyoungjune Pak, Seong-Jang Kim, In Joo Kim, Bo Hyun Kim, Sang Soo Kim, and Yun Kyung Jeon
The incidence of thyroid cancer in both men and women is increasing faster than that of any other cancer. Although positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) has received much attention, the use of FDG PET for the management of thyroid cancer is limited primarily to postoperative follow-up. However, it might have a role in selected, more aggressive pathologies, and so patients at a high risk of distant metastasis may benefit from PET before surgery. As less FDG-avid thyroid cancers may lower the diagnostic accuracy of PET in preoperative assessment, an understanding of FDG avidity is important for the evaluation of thyroid cancer. FDG avidity has been shown to be associated with tumor size, lymph node metastasis, and glucose transporter expression and differentiation. As PET is commonly used in clinical practice, the detection of incidentalomas by PET is increasing. However, incidentalomas detected by PET have a high risk of malignancy. Clinicians handling cytologically indeterminate nodules face a dilemma regarding a procedure for a definitive diagnosis, usually lobectomy. With ‘nondiagnostic (ND)’ fine-needle biopsy (FNA), PET has shown a negative predictive value (NPV) of 100%, which indicates that negative uptake in a ND FNA procedure accurately excludes malignancy. With ‘atypia of undetermined significance’ or ‘follicular neoplasm’, the sensitivity and NPV of PET are 84 and 88%. PET does not provide additional information for the preoperative assessment of thyroid cancer. However, factors associated with FDG positivity are related to a poor prognosis; therefore, FDG PET scans before surgery may facilitate the prediction of the prognosis of differentiated thyroid cancer.
Muhammad Yasir Asghar, Tero Viitanen, Kati Kemppainen, and Kid Törnquist
Anaplastic thyroid cancer (ATC) is the most aggressive form of human thyroid cancer, lacking any effective treatment. Sphingosine 1-phosphate (S1P) receptors and human ether-a′-go-go-related gene (HERG (KCNH2)) potassium channels are important modulators of cell migration. In this study, we have shown that the S1P1–3 receptors are expressed in C643 and THJ-16T human ATC cell lines, both at mRNA and protein level. S1P inhibited migration of these cells and of follicular FTC-133 thyroid cancer cells. Using the S1P1,3 inhibitor VPC-23019, the S1P2 inhibitor JTE-013, and the S1P2 receptor siRNA, we showed that the effect was mediated through S1P2. Treatment of the cells with the Rho inhibitor C3 transferase abolished the effect of S1P on migration. S1P attenuated Rac activity, and inhibiting Rac decreased migration. Sphingosine kinase inhibitor enhanced basal migration of cells, and addition of exogenous S1P inhibited migration. C643 cells expressed a nonconducting HERG protein, and S1P decreased HERG protein expression. The HERG blocker E-4031 decreased migration. Interestingly, downregulating HERG protein with siRNA decreased the basal migration. In experiments using HEK cells overexpressing HERG, we showed that S1P decreased channel protein expression and current and that S1P attenuated migration of the cells. We conclude that S1P attenuates migration of C643 ATC cells by activating S1P2 and the Rho pathway. The attenuated migration is also, in part, dependent on a S1P-induced decrease of HERG protein.
Maria Rosaria Sapio, Anna Guerra, Daniela Posca, Paolo Piero Limone, Maurilio Deandrea, Manuela Motta, Giancarlo Troncone, Alessia Caleo, Pasquale Vallefuoco, Guido Rossi, Gianfranco Fenzi, and Mario Vitale
Ten to fifteen percent of fine-needle aspiration biopsy (FNAB) of thyroid nodules are indeterminate. Galectin-3 (Gal-3) and the oncogene BRAF V600E are markers of malignancy useful to improve FNAB accuracy. The objective of this study was to determine whether the combined analysis of Gal-3 and BRAF V600E expression in thyroid aspirates could improve the diagnosis in FNAB with suspicious cytological findings. Two hundred and sixty-one surgical thyroid tissues and one hundred and forty-four thyroid aspirates were analyzed for the presence of the two markers. In surgical specimens, Gal-3 expression was present in 27.4% benign nodules, 91.9% papillary (PTC) and 75% follicular (FTC) thyroid carcinomas. BRAF V600E was not detected in 127 benign nodules, as well as in 32 FTCs, while was found in 42.9% PTC. No correlation was found between BRAF mutation and Gal-3 expression. Forty-seven consecutive FNAB suspicious for PTC were analyzed for the presence of the two markers. Of these nodules, 23 were benign at histology, 6 were positive for Gal-3, none displayed BRAF V600E, and 17 were negative for both the markers. Twenty suspicious nodules were diagnosed as PTC and four FTCs at histology. Of these 24 carcinomas, 9 resulted positive for BRAF V600E, 17 for Gal-3, and 22 for one or both the markers. The sensitivity, specificity, and accuracy for the presence of Gal-3 and/or BRAFV600E were significantly higher than those obtained for the two markers alone. Notably, the negative predictive value increased from 70.8 to 89.5%. In conclusion, the combined detection of Gal-3 and BRAF V600E improves the diagnosis in FNAB with cytological findings suspicious for PTC and finds clinical application in selected cases.
Dara O Kavanagh, Marie McIlroy, Eddie Myers, Fiona Bane, Thomas B Crotty, E McDermott, Arnold D Hill, and Leonie S Young
Epidemiological, clinical, and molecular studies suggest a role for oestrogen in thyroid cancer. How oestrogen mediates its effects and the consequence of it on clinical outcome has not been fully elucidated. The participation of coregulatory proteins in modulating oestrogen receptor (ER) function and input of crosstalk with the tyrosine kinase receptor HER2 was investigated. Oestrogen induced cell proliferation in the follicular thyroid cancer (FTC)-133 cells, but not in the anaplastic 8305C cell line. Knockdown of the coactivator steroid receptor coactivator (SRC)-1 inhibited FTC-133 basal, but not oestrogen induced, cell proliferation. Oestrogen also increased protein expression of SRC-1 and the ER target gene cyclin D1 in the FTC-133 cell line. ERα, ERβ, the coregulatory proteins SRC-1 and nuclear corepressor (NCoR), and the tyrosine kinase receptor HER2 were localised by immunohistochemistry and immnofluorescence in paraffin-embedded tissue from thyroid tumour patients (n=111). ERα was colocalised with both SRC-1 and NCoR to the nuclei of the tumour epithelial cells. Expression of ERα and NCoR was found predominantly in non-anaplastic tumours and was significantly associated with well-differentiated tumours and reduced incidence of disease recurrence. In non-anaplastic tumours, HER2 was significantly associated with SRC-1, and these proteins were associated with poorly differentiated tumours, capsular invasion and disease recurrence. Totally, 87% of anaplastic tumours were positive for SRC-1. Kaplan–Meier estimates of disease-free survival indicated that in thyroid cancer, SRC-1 strongly correlates with reduced disease-free survival (P<0.001), whereas NCoR predicted increased survival (P<0.001). These data suggest opposing roles for the coregulators SRC-1 and NCoR in thyroid tumour progression.
Alessandro Antonelli, Silvia Martina Ferrari, Poupak Fallahi, Silvia Frascerra, Simona Piaggi, Stefania Gelmini, Cristiana Lupi, Michele Minuto, Piero Berti, Salvatore Benvenga, Fulvio Basolo, Claudio Orlando, and Paolo Miccoli
In papillary thyroid carcinomas (PTCs), oncogenes activate a transcriptional program including the upregulation of CXCL10 chemokine, which stimulates proliferation and invasion. Furthermore, peroxisome proliferator-activated receptor-γ (PPARγ) activators thiazolidinediones (TZDs) modulate CXCL10 secretion in normal thyroid follicular cells (TFC), and inhibit PTC growth. Until now, no study has evaluated the effect of cytokines on CXCL10 secretion in PTCs, nor the effect of PPARγ activation. The combined effects of interferon γ (IFNγ) and tumor necrosis factor α (TNFα) stimulation on CXCL10 secretion in primary cells from PTCs and TFC were tested. Furthermore, the effect of PPARγ activation by TZDs, on CXCL10 secretion and proliferation in these cell types was studied. In primary cultures of TFC and PTCs CXCL10 production was absent under basal conditions; a similar dose-dependent secretion of CXCL10 was induced by IFNγ in both cell types. TNFα alone induced a slight but significant CXCL10 secretion only in PTCs. The stimulation with IFNγ+TNFα induced a synergistic CXCL10 release in both cell types; however, a secretion more than ten times higher was induced in PTCs. Treatment of TFC with TZDs dose-dependently suppressed IFNγ+TNFα-induced CXCL10 release, while TZDs stimulated CXCL10 secretion in PTCs. A significant antiproliferative effect by TZDs was observed only in PTCs. In conclusion, a dysregulation of CXCL10 secretion has been shown in PTCs. In fact, a CXCL10 secretion more than ten times higher has been induced by IFNγ+TNFα in PTCs with respect to TFC. Moreover, TZDs inhibited CXCL10 secretion in TFC and stimulated it in PTCs. The effect of TZDs on CXCL10 was unrelated to the significant antiproliferative effect in PTCs.
Eleonore Fröhlich, Fausto Machicao, and Richard Wahl
Differentiating drugs may be able to re-sensitize thyroid carcinomas to radioiodine therapy. Substituted thiazolidinediones (TZDs) belong to the group of oral anti-diabetic drugs that also possess anti-proliferative and pro-apoptotic effects and, potentially, differentiating effects on several cancer cell lines. Some of the effects are mediated via the peroxisome proliferator-activated receptor gamma (PPAR-γ). We investigated the effect of troglitazone, rosiglitazone and pioglitazone on differentiation in normal porcine thyrocytes and in the follicular carcinoma cell lines FTC 133 and FTC 238. Differentiation was investigated by measuring 125I uptake and the expression of sodium-iodide symporter and thyroglobulin proteins. The TZDs were tested in the presence of retinol and retinoic acid. Additionally, proliferation was evaluated by [3H]thymidine uptake and cell number and apoptosis by annexin V-labeling. Controls included tocopherol and unsubstituted thiazolidinedione and co-incubation of the TZDs with the PPAR-γ antagonist GW9662. PPAR-γ and retinol X receptor (RXR)-α were investigated by immunocytochemistry, Western blot and RT-PCR. Cells derived from the metastasis showed greater responses than cells derived from the primary tumor. Troglitazone showed greater effects than the other TZDs. Troglitazone significantly increased 125I uptake and apoptosis and decreased [3H]thymidine uptake and cell number. The amount of the sodium iodide-symporter in the membrane fraction was significantly increased, while that of thyroglobulin was not influenced by the treatment. Inclusion of antagonist did not abolish these effects. No synergistic effect with any retinoid was detected. All transformed cells expressed PPAR-γ and RXR-α but TZDs did not change their expression.
Troglitazone appears to be suited for the re-differentiation treatment of dedifferentiated thyroid carcinoma because its action is twofold. On the one hand it increases differentiation and on the other hand it inhibits proliferation.
J Lado-Abeal, R Celestino, S B Bravo, M E R Garcia-Rendueles, J de la Calzada, I Castro, P Castro, C Espadinha, F Palos, P Soares, C V Alvarez, M Sobrinho-Simões, and J Cameselle-Teijeiro
Our main objective was to search for mutations in candidate genes and for paired box gene 8–peroxisome proliferator-activated receptor gamma (PAX8–PPARγ) rearrangement in a well-differentiated angioinvasive follicular thyroid carcinoma (FTC) causing hyperthyroidism. DNA and RNA were extracted from the patient's thyroid tumor, as well as ‘normal’ thyroid tissue, and from peripheral blood lymphocytes (PBLs) of the patient, her daughter, and two siblings. Nuclear isolation was extracted from the patient's tumor, ’normal’ thyroid tissue, PBLs, and uterine leiomyoma tissue. TSH receptor (TSHR), RAS, and BRAF genes were sequenced. We searched for PAX8 – PPAR γ in thyroid, PBL, and uterine leiomyoma samples from the patient and family members. Proliferative effects of detected mutants on non-transformed human thyrocytes cultures. An activating TSHR mutation, M453T, was detected in the tumor. PAX8 (exons 1 – 8 + 10)– PPAR γ was found in all tested patient's tissues. A second rearrangement, PAX8 (exons 1 – 8)– PPAR γ, was detected in the patient's normal thyroid tissue. Under deprived medium condition, co-transfection of PAX8 – PPAR γ and TSHR – M453T dramatically increased the number of thyrocytes, an effect that it was not observed with TSHR wild-type (WT); under complete medium conditions, co-transfection of PAX8 – PPAR γ with either TSHR – M453T or TSHR – WT inhibited cell proliferation. We report a patient with hyperthyroidism due to a FTC bearing an activating TSHR mutation and PAX8 – PPAR γ rearrangements. PAX8 – PPAR γ was present as a mosaicism affecting tissues from endodermal and mesodermal origin. PAX8 – PPAR γ and TSHR – M453T inhibited or promoted thyrocyte proliferation depending on medium conditions. The activating TSHR mutation could promote in vivo FTC development in PAX8 – PPAR γ-positive thyrocytes under poor blood supply with deprivation of growth factors but restraint the tumor growth when growth factors are supplied.
Hai-Yan Zhang, Hua-Qin Wang, Hai-Mei Liu, Yifu Guan, and Zhen-Xian Du
DJ-1, a cancer-associated protein protects cells from multiple toxic stresses. The expression of DJ-1 and its influence on thyroid cancer cell death has not been investigated so far. We analyzed DJ-1 expression in human thyroid carcinoma cell lines and the effect of DJ-1 on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. DJ-1 was expressed in human thyroid carcinoma cell lines; small interfering RNA-mediated downregulation of its levels significantly sensitized thyroid carcinoma cells to TRAIL-induced apoptosis, whereas the forced exogenous expression of DJ-1 significantly suppressed cell death induced by TRAIL. We also report here that TRAIL-induced thyroid cancer cell apoptosis is mediated by oxidative stress and that DJ-1, a potent nutritional antioxidant, protects cancer cells from apoptosis at least in part by impeding the elevation of reactive oxygen species levels induced by TRAIL and impairing caspase-8 activation. Subsequently, we investigated DJ-1 expression in 52 normal and 74 primary thyroid carcinomas from patients of China Medical University. The protein was not detectable in the 52 specimens of normal thyroid, while 70 out of 74 analyzed carcinomas (33 out of 33 follicular, 17 out of 19 papillary, 12 out of 13 medullar, and 8 out of 9 anaplastic) were clearly positive for DJ-1 expression. Our data demonstrated that DJ-1 is specifically expressed in thyroid carcinomas and not in the normal thyroid tissue. In addition, the protein modulates the response to TRAIL-mediated apoptosis in human neoplastic thyroid cells, at least partially through its antioxidant property.