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.
Muhammad Yasir Asghar, Tero Viitanen, Kati Kemppainen, and Kid Törnquist
Xiulong Xu, Helen Ding, Geetha Rao, Shalini Arora, Constantine P Saclarides, Joseph Esparaz, Paolo Gattuso, Carmen C Solorzano, and Richard A Prinz
The sonic hedgehog (SHH) pathway is activated in several types of malignancy and plays an important role in tumor cell proliferation and tumorigenesis. SHH binding to a 12-pass transmembrane receptor, Patched (PTCH), leads to freeing of Smoothened (SMO) and subsequent activation of GLI transcription factors. In the present study, we analyzed the expression of SHH, PTCH, SMO, and GLI1 in 31 follicular thyroid adenomas (FTA), 8 anaplastic thyroid carcinomas (ATC), and 51 papillary thyroid carcinomas (PTC) by immunohistochemical staining. More than 65% of FTA, PTC, and ATC specimens stained positive for SHH, PTCH, SMO, and GLI. However, the expression of the genes encoding these four molecules did not correlate with any clinicopathologic parameters, including the age, gender, the status of BRAF gene mutation, tumor stage, local invasion, and metastasis. Three thyroid tumor cell lines (KAT-18, WRO82, and SW1736) all expressed the genes encoding these four molecules. 5-Bromo-2-deoxyuridine labeling and MTT cell proliferation assays revealed that cyclopamine (CP), an inhibitor of the SHH pathway, was able to inhibit the proliferation of KAT-18 and WRO82 cells more effectively than SW1736 cells. CP led to the arrest of cell cycle or apoptosis. Knockdown of SHH and GLI expression by miRNA constructs that target SHH or GLI mRNA in KAT-18 and SW1736 cells led to the inhibition of cell proliferation. Our results suggest that the SHH pathway is widely activated in thyroid neoplasms and may have potential as an early marker of thyroid cancer or as a potential therapeutic target for thyroid cancer treatment.
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.
Kirk Jensen, Athanasios Bikas, Aneeta Patel, Yevgeniya Kushchayeva, John Costello, Dennis McDaniel, Kenneth Burman, and Vasyl Vasko
The HIV protease inhibitor Nelfinavir (NFV) inhibits PI3K/AKT and MAPK/ERK signaling pathways, emerging targets in thyroid cancers. We examined the effects of NFV on cancer cells that derived from follicular (FTC), papillary (PTC) and anaplastic (ATC) thyroid cancers. NFV (1–20 µM) was tested in FTC133, BCPAP and SW1736 cell lines. The effects of NFV on cell proliferation were determined in vitro using real-time microscopy and by flow cytometry. DNA damage, apoptotic cell death and expression of molecular markers of epithelial–mesenchymal transition (EMT) were determined by Western blot and real-time PCR. Real-time imaging demonstrated that NFV (10 µM) increased the time required for the cell passage through the phases of cell cycle and induced DNA fragmentation. Growth inhibitory effects of NFV were associated with the accumulation of cells in G0/G1 phase, downregulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). NFV also induced the expression of γH2AX and p53BP1 indicating DNA damage. Treatment with NFV (20 µM) resulted in caspase-3 cleavage in all examined cells. NFV (20 µM) decreased the levels of total and p-AKT in PTEN-deficient FTC133 cells. NFV had no significant effects on total ERK and p-ERK in BRAF-positive BCPAP and SW1736 cells. NFV had no effects on the expression of EMT markers (Twist, Vimentin, E- and N-Cadherin), but inhibited the migration and decreased the abilities of thyroid cancer cells to survive in non-adherent conditions. We conclude that NFV inhibits proliferation and induces DNA damage in thyroid cancer cell lines. Our in vitro data suggest that NFV has a potential to become a new thyroid cancer therapeutic agent.
Ying Ni, Spencer Seballos, Shireen Ganapathi, Danielle Gurin, Benjamin Fletcher, Joanne Ngeow, Rebecca Nagy, Richard T Kloos, Matthew D Ringel, Thomas LaFramboise, and Charis Eng
Along with breast and endometrial cancers, thyroid cancer is a major component cancer in Cowden syndrome (CS). Germline variants in SDHB/C/D (SDHx) genes account for subsets of CS/CS-like cases, conferring a higher risk of breast and thyroid cancers over those with only germline PTEN mutations. To investigate whether SDHx alterations at both germline and somatic levels occur in apparently sporadic breast cancer and differentiated thyroid cancer (DTC), we analyzed SDHx genes in the following four groups: i) 48 individuals with sporadic invasive breast adenocarcinoma for germline mutation; ii) 48 (expanded to 241) DTC for germline mutation; iii) 37 pairs DTC tumor-normal tissues for germline and somatic mutation and mRNA expression levels; and iv) data from 476 patients in the Cancer Genome Atlas thyroid carcinoma dataset for validation. No germline SDHx variant was found in a pilot series of 48 breast cancer cases. As germline SDHx variants were found in our pilot of 48 thyroid cancer cases, we expanded to three series of DTC comprising a total 754 cases, and found 48 (6%) with germline SDHx variants (P<0.001 compared with 0/350 controls). In 513 tumors, we found 27 (5%) with large somatic duplications within chromosome 1 encompassing SDHC. Both papillary and follicular thyroid tumors showed consistent loss of SDHC/D gene expression (P<0.001), which is associated with earlier disease onset and higher pathological-TNM stage. Therefore, we conclude that both germline and somatic SDHx mutations/variants occur in sporadic DTC but are very rare in sporadic breast cancer, and overall loss of SDHx gene expression is a signature of DTC.
C Passler, C Scheuba, G Prager, K Kaczirek, K Kaserer, G Zettinig, and B Niederle
Papillary (PTC) and follicular thyroid carcinoma (FTC) are known as differentiated thyroid carcinoma (DTC). Nevertheless, according to the UICC/AJCC (TNM) classification PTC and FTC are frequently analyzed as one cancer. The aim of this study is to show differences in outcome and specific prognostic factors in an iodine-replete endemic goiter region. Six hundred and three patients with DTC treated within a 35-year-period were retrospectively analyzed with respect to carcinoma-specific survival. Prognostic factors were tested for their significance using univariate and multivariate analysis. The histological type (PTC versus FTC) was found to be a highly significant factor - carcinoma-specific survival both in univariate (P<0.001) and multivariate analyses (P=0.003) was significantly different. Univariate analysis revealed patients' age, extra-thyroid tumor spread, lymph node and distant metastases, increasing tumor size, and the tall cell variant to be significant prognostic factors for PTC patients. Age > or =45 years, positive lymph nodes and increasing tumor size were confirmed as independent prognostic factors. Univariate analysis of FTC patients revealed age at presentation, gender, extrathyroidal tumor spread, lymph node and distant metastases, increasing tumor size, multifocality, widely invasive tumor growth and oxyphilic variant to be factors bearing prognostic significance. The presence of distant metastases and increasing tumor size could be identified as independent prognostic factors for FTC patients. This study shows distinctive differences in prognostic factors of PTC and FTC: independent factors predicting poor prognosis are age > or =45 years, positive lymph nodes and increasing tumor size for PTC, and distant metastases and increasing tumor size for FTC. PTC and FTC patients should be analyzed and reported separately.
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.
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.
D Deandreis, A Al Ghuzlan, S Leboulleux, L Lacroix, J P Garsi, M Talbot, J Lumbroso, E Baudin, B Caillou, J M Bidart, and M Schlumberger
The aim of this study is to search for relationships between histology, radioiodine (131I) uptake, fluorodeoxyglucose (FDG) uptake, and disease outcome in patients with metastatic thyroid cancer. Eighty patients with metastatic thyroid cancer (34 males, 46 females, mean age at the time of the diagnosis of metastases: 55 years) were retrospectively studied. All patients were treated with radioactive iodine and evaluated by FDG-positron emission tomography (PET). Primary tumor tissue sample was available in all cases. Forty-five patients (56%) had a papillary, 12 (15%) a follicular, and 23 (29%) a poorly differentiated thyroid cancer. Cellular atypias, necrosis, mitoses, thyroid capsule infiltration, and vascular invasion were frequently detected (70, 44, 52, 60, and 71% respectively). Metastases disclosed FDG uptake in 58 patients (72%) and 131I uptake in 37 patients (45%). FDG uptake was the only significant prognostic factor for survival (P=0.02). The maximum standardized uptake value and the number of FDG avid lesions were also related to prognosis (P=0.03 and 0.009). Age at the time of the diagnosis of metastases (P=0.001) and the presence of necrosis (P=0.002) were independent predictive factors of FDG uptake. Radioiodine uptake was prognostic for stable disease (P=0.001) and necrosis for progressive disease at 1 year (P=0.001). Histological subtype was not correlated with in vivo tumor metabolism and prognosis. In conclusion, FDG uptake in metastatic thyroid cancer is highly prognostic for survival. Histological subtype alone does not correlate with 131I/FDG uptake pattern and patient outcome. Well-differentiated thyroid cancer presenting histological features such as necrosis and FDG uptake on PET scan should be considered aggressive differentiated cancers.