Peroxisome proliferator-activated receptor γ (PPARγ) has been demonstrated to be anti-neoplastic against various human tumors. The aim of this study was to delineate the molecular mechanism underlying PPARγ ligand rosiglitazone (BRL) antiproliferative effects in follicular WRO and anaplastic FRO human thyroid carcinoma cells. BRL upregulated the p21Cip1/WAF1 levels in the two thyroid cancer cells, while did not modify the p53 protein content. Different evidences indicate that the p21Cip1/WAF1 upregulation by BRL requires a functional PPARγ, since it was reversed by silencing PPARγ and pretreatment with GW9662, an irreversible PPARγ antagonist. Transient transfection assays showed that BRL triggered the transcriptional activity of p21Cip1/WAF1 promoter gene in a p53-independent way, being a p21Cip1/WAF1 promoter construct deleted in the p53 sites still activated by BRL. The Sp1 inhibitor mithramycin silenced the p21Cip1/WAF1 promoter activity suggesting an important role of Sp1 in mediating BRL activation. The electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) assays evidenced a functional interaction between PPARγ and Sp1 in regulating p21Cip1/WAF1. Intriguingly, ChIP analysis revealed in the p21Cip1/WAF1 gene promoter an increased recruitment of the RNA Pol II associated with an increased histone H3 acetylation and a reduced H3 methylation. The biological event, consistent with PPARγ-induced WRO and FRO cell growth inhibition, was reversed by p21Cip1/WAF1 antisense oligonucleotides and was confirmed by increasing the PPARγ expression, suggesting a crucial role exerted by p21Cip1/WAF1 in PPARγ action. Our results further candidate BRL as a potential agent able to inhibit tumor progression of follicular and anaplastic thyroid carcinoma.
D Bonofiglio, H Qi, S Gabriele, S Catalano, S Aquila, M Belmonte, and S Andò
Kirk Jensen, Aneeta Patel, Joanna Klubo-Gwiezdzinska, Andrew Bauer, and Vasyl Vasko
Resistance to anoikis (matrix deprivation-induced apoptosis) is a critical component of the metastatic cascade. Molecular mechanisms underlying resistance to anoikis have not been reported in thyroid cancer cells. For an in vitro model of anoikis, we cultured follicular, papillary, and anaplastic thyroid cancer cell lines on poly-HEMA-treated low-adherent plates. We also performed immunohistochemical analysis of human cancer cells that had infiltrated blood and/or lymphatic vessels. Matrix deprivation was associated with establishment of contacts between floating thyroid cancer cells and formation of multi-cellular spheroids. This process was associated with activation of gap junctional transfer. Increased expression of the gap junction molecule Connexin43 was found in papillary and anaplastic cancer cells forming spheroids. All non-adherent cancer cells showed a lower proliferation rate compared with adherent cells but were more resistant to serum deprivation. AKT was constitutively activated in cancer cells forming spheroids. Inhibition of gap junctional transfer through Connexin43 silencing, or by treatment with the gap junction disruptor carbenoxolone, resulted in loss of pAKT and induction of apoptosis in a cell-type-specific manner. In human thyroid tissue, cancer cells that had infiltrated blood vessels showed morphological similarity to cancer cells forming spheroids in vitro. Intra-vascular cancer cells demonstrated prominent AKT activation in papillary and follicular cancers. Increased Connexin43 immunoreactivity was observed only in intra-vascular papillary cancer cells. Our data demonstrate that establishment of inter-cellular communication contributes to thyroid cancer cell resistance to anoikis. These findings suggest that disruption of gap junctional transfer could represent a potential therapeutic strategy for prevention of metastases.
J D Lin, M J Liou, T C Chao, H F Weng, and Y S Ho
From 1977 through 1995, 1,013 thyroid carcinoma patients received treatment and were followed up at Chang Gung Medical Center in Taiwan. To evaluate the prognostic variables of papillary and follicular thyroid carcinomas with limited lymph node metastases, a retrospective review of these patients was performed. Of these patients, 910 had papillary or follicular thyroid carcinoma, and 119 patients were categorized as clinical stage 2 with limited neck lymph node metastases only at the time of diagnosis. The patients were categorized into two groups as no recurrence and local recurrence or distant metastasis at the end of 1997. After the operations, radioactive iodide (131I) treatments were performed in 114 patients and external radiotherapy for neck region or distant metastases in 18 patients. The median follow-up period of these patients was 5.4 years. Clinical variables were coded in our computer for statistical analysis. After the treatments, 93 patients remained disease-free; 10 were in stage 2; 5 in stage 3; and 11 aggravated to stage 4. Of the clinical variables, age, post-operative first 1311 uptake scans, and 1-month post-operative thyroglobulin levels revealed statistically significant differences between the group which improved and the group which did not. During the follow-up period, five patients died; three patients died of thyroid cancer and two died of intercurrent diseases. Patients with papillary thyroid carcinoma revealed a higher percentage of lymph node metastases. Although limited lymph node metastases did not influence survival rate, patients with poor prognostic factors need more aggressive treatment to avoid progression of the cancer.
Xiaoli Liu, Justin Bishop, Yuan Shan, Sara Pai, Dingxie Liu, Avaniyapuram Kannan Murugan, Hui Sun, Adel K El-Naggar, and Mingzhao Xing
Mutations 1 295 228 C>T and 1 295 250 C>T (termed C228T and C250T respectively), corresponding to −124 C>T and −146 C>T from the translation start site in the promoter of the telomerase reverse transcriptase (TERT) gene, have recently been reported in human cancers, but not in thyroid cancers yet. We explored these mutations in thyroid cancers by genomic sequencing of a large number of primary tumor samples. We found the C228T mutation in 0 of 85 (0.0%) benign thyroid tumors, 30 of 257 (11.7%) papillary thyroid cancers (PTC), 9 of 79 (11.4%) follicular thyroid cancers (FTC), 3 of 8 (37.5%) poorly differentiated thyroid cancers (PDTC), 23 of 54 (42.6%) anaplastic thyroid cancers (ATC), and 8 of 12 (66.7%) thyroid cancer cell lines. The C250T mutation was uncommon, but mutually exclusive with the C228T mutation, and the two mutations were collectively found in 11 of 79 (13.9%) FTC, 25 of 54 (46.3%) ATC, and 11 of 12 (91.7%) thyroid cancer cell lines. Among PTC variants, the C228T mutation was found in 4 of 13 (30.8%) tall-cell PTC (TCPTC), 23 of 187 (12.3%) conventional PTC, and 2 of 56 (3.6%) follicular variant PTC samples. No TERT mutation was found in 16 medullary thyroid cancer samples. The C228T mutation was associated with the BRAF V600E mutation in PTC, being present in 19 of 104 (18.3%) BRAF mutation-positive PTC vs 11 of 153 (7.2%) the BRAF mutation-negative PTC samples (P=0.0094). Conversely, BRAF mutation was found in 19 of 30 (63.3%) C228T mutation-positive PTC vs 85 of 227 (37.4%) C228T mutation-negative PTC samples (P=0.0094). We thus for the first time, to our knowledge, demonstrate TERT promoter mutations in thyroid cancers, that are particularly prevalent in the aggressive thyroid cancers TCPTC, PDTC, ATC and BRAF mutation-positive PTC, revealing a novel genetic background for thyroid cancers.
J M Gómez, N Gómez, M Sahún, A Rafecas, C Villabona, and J Soler
Despite the usual excellent prognosis of differentiated thyroid carcinoma, some patients die because of disease. It has been speculated that lethal disease may have a better prognosis if patients are treated with extensive surgery plus 131I ablative treatment. We have analyzed a group of 223 patients with differentiated thyroid carcinoma treated under a uniform therapeutic protocol of surgery and followed for 3 to 17.7 years, in order to differentiate patients with a high and a low risk of mortality and the influence of therapy on survival rate.
The therapeutic protocol was as follows. If the diagnosis was papillary carcinoma, subtotal thyroidectomy was performed and cervical nodes were removed if they were suspicious for cancer. If the diagnosis was follicular carcinoma, a total thyroidectomy was performed. 131I was given in cases of patients who were more than 60 years old or who had extrathyroid disease or metastases in papillary carcinomas and in macroangioinvasive follicular carcinomas. In survival analysis, the event used as the end-point was death due to thyroid carcinoma and summarized by the Kaplan-Meier curve and the Mantel-Cox method.
We found three independent prognostic factors which determined mortality: over 60 years of age, tumor size larger than 6 cm and metastases. On the basis of these factors we identified two risk groups: a low-risk group (A), who had no risk factors, composed of 153 patients whose survival rate at 205 months was 100% and a high-risk group (B), who had one or more risk factors, composed of 55 patients whose survival rate at 213 months was 39.6%. Seventeen patients in this second group died from thyroid carcinoma. We therefore analyzed the effect of treatment in group B. Patients who had more extensive surgery had a similar survival rate to those who had less extensive surgery and 131I administration did not modify the survival rate.
These data support the idea that the identification of low-risk groups may facilitate a more rational approach to treatment of differentiated thyroid carcinoma, avoiding aggressive therapy in cases with a good prognosis.
Endocrine-Related Cancer (1997) 4 459-464
Aruna V Krishnan and David Feldman
Calcitriol, the hormonally active form of vitamin D, exerts multiple anti-proliferative and pro-differentiating actions including cell cycle arrest and induction of apoptosis in many malignant cells, and the hormone is currently being evaluated in clinical trials as an anti-cancer agent. Recent research reveals that calcitriol also exhibits multiple anti-inflammatory effects. First, calcitriol inhibits the synthesis and biological actions of pro-inflammatory prostaglandins (PGs) by three mechanisms: i) suppression of the expression of cyclooxygenase-2, the enzyme that synthesizes PGs; ii) up-regulation of the expression of 15-hydroxyprostaglandin dehydrogenase, the enzyme that inactivates PGs; and iii) down-regulation of the expression of PG receptors that are essential for PG signaling. The combination of calcitriol and nonsteroidal anti-inflammatory drugs results in a synergistic inhibition of the growth of prostate cancer (PCa) cells and offers a potential therapeutic strategy for PCa. Second, calcitriol increases the expression of mitogen-activated protein kinase phosphatase 5 in prostate cells resulting in the subsequent inhibition of p38 stress kinase signaling and the attenuation of the production of pro-inflammatory cytokines. Third, calcitriol also exerts anti-inflammatory activity in PCa through the inhibition of nuclear factor-κB signaling that results in potent anti-inflammatory and anti-angiogenic effects. Other important direct effects of calcitriol as well as the consequences of its anti-inflammatory effects include the inhibition of tumor angiogenesis, invasion, and metastasis. We hypothesize that these anti-inflammatory actions, in addition to the other known anti-cancer effects of calcitriol, play an important role in its potential use as a therapeutic agent for PCa. Calcitriol or its analogs may have utility as chemopreventive agents and should be evaluated in clinical trials in PCa patients with early or precancerous disease.
Catherine Ory, Nicolas Ugolin, Céline Levalois, Ludovic Lacroix, Bernard Caillou, Jean-Michel Bidart, Martin Schlumberger, Ibrahima Diallo, Florent de Vathaire, Paul Hofman, José Santini, Bernard Malfoy, and Sylvie Chevillard
Both external and internal exposure to ionizing radiation are strong risk factors for the development of thyroid tumors. Until now, the diagnosis of radiation-induced thyroid tumors has been deduced from a network of arguments taken together with the individual history of radiation exposure. Neither the histological features nor the genetic alterations observed in these tumors have been shown to be specific fingerprints of an exposure to radiation. The aim of our work is to define ionizing radiation-related molecular specificities in a series of secondary thyroid tumors developed in the radiation field of patients treated by radiotherapy. To identify molecular markers that could represent a radiation-induction signature, we compared 25K microarray transcriptome profiles of a learning set of 28 thyroid tumors, which comprised 14 follicular thyroid adenomas (FTA) and 14 papillary thyroid carcinomas (PTC), either sporadic or consecutive to external radiotherapy in childhood. We identified a signature composed of 322 genes which discriminates radiation-induced tumors (FTA and PTC) from their sporadic counterparts. The robustness of this signature was further confirmed by blind case-by-case classification of an independent set of 29 tumors (16 FTA and 13 PTC). After the histology code break by the clinicians, 26/29 tumors were well classified regarding tumor etiology, 1 was undetermined, and 2 were misclassified. Our results help shed light on radiation-induced thyroid carcinogenesis, since specific molecular pathways are deregulated in radiation-induced tumors.
Urbain Weyemi, Bernard Caillou, Monique Talbot, Rabii Ameziane-El-Hassani, Ludovic Lacroix, Odile Lagent-Chevallier, Abir Al Ghuzlan, Dirk Roos, Jean-Michel Bidart, Alain Virion, Martin Schlumberger, and Corinne Dupuy
NADPH oxidase 4 (NOX4) belongs to the NOX family that generates reactive oxygen species (ROS). Function and tissue distribution of NOX4 have not yet been entirely clarified. To date, in the thyroid gland, only DUOX1/2 NOX systems have been described. NOX4 mRNA expression, as shown by real-time PCR, was present in normal thyroid tissue, regulated by TSH and significantly increased in differentiated cancer tissues. TSH increased the protein level of NOX4 in human thyroid primary culture and NOX4-dependent ROS generation. NOX4 immunostaining was detected in normal and pathologic thyroid tissues. In normal thyroid tissue, staining was heterogeneous and mostly found in activated columnar thyrocytes but absent in quiescent flat cells. Papillary and follicular thyroid carcinomas displayed more homogeneous staining. The p22phox protein that forms a heterodimeric enzyme complex with NOX4 displayed an identical cellular expression pattern and was also positively regulated by TSH. ROS may have various biological effects, depending on the site of production. Intracellular NOX4–p22phox localization suggests a role in cytoplasmic redox signaling, in contrast to the DUOX localization at the apical membrane that corresponds to an extracellular H2O2 production. Increased NOX4–p22phox in cancer might be related to a higher proliferation rate and tumor progression but a role in the development of tumors has to be further studied and established in the future.
M M Muresan, P Olivier, J Leclère, F Sirveaux, L Brunaud, M Klein, R Zarnegar, and G Weryha
The presence of distant metastases from differentiated thyroid carcinoma decreases the 10-year survival of patients by 50%. Bone metastases represent a frequent complication especially of follicular thyroid cancer and severely reduce the quality of life causing pain, fractures, and spinal cord compression. Diagnosis is established by correlating clinical suspicion with imaging. Imaging is essential to detect, localize, and assess the extension of the lesions and should be used in conjunction with clinical evidence. Bone metastases are typically associated with elevated markers of bone turnover, but these markers have not been evaluated in differentiated thyroid cancer. Skeletal and whole-body magnetic resonance imaging and fusion 2-deoxy-2-[18F]fluoro-d-glucose whole-body positron emission tomography/computed tomography (PET/CT) are the best anatomic and functional imaging techniques available in specialized centers. For well-differentiated lesions, iodine-PET scan combined 124I-PET/CT is the newest imaging development and 131I is the first line of treatment. Bisphosphonates reduce the complications rate and pain, alone or in combination with radioiodine, radionuclides, or external beam radiotherapy and should be employed. Surgery and novel minimally invasive consolidation techniques demand an appropriate patient selection for best results on a multimodal approach. Basic research on interactions between tumor cells and bone microenvironment are identifying potential novel targets for future more effective therapeutic interventions for less differentiated tumors.
Roberto Bellelli, Maria Domenica Castellone, Ginesa Garcia-Rostan, Clara Ugolini, Carmelo Nucera, Peter M Sadow, Tito Claudio Nappi, Paolo Salerno, Maria Carmela Cantisani, Fulvio Basolo, Tomas Alvarez Gago, Giuliana Salvatore, and Massimo Santoro
Anaplastic thyroid carcinoma (ATC) is a very aggressive thyroid cancer. forkhead box protein M1 (FOXM1) is a member of the forkhead box family of transcription factors involved in control of cell proliferation, chromosomal stability, angiogenesis, and invasion. Here, we show that FOXM1 is significantly increased in ATCs compared with normal thyroid, well-differentiated thyroid carcinomas (papillary and/or follicular), and poorly differentiated thyroid carcinomas (P=0.000002). Upregulation of FOXM1 levels in ATC cells was mechanistically linked to loss-of-function of p53 and to the hyperactivation of the phosphatidylinositol-3-kinase/AKT/FOXO3a pathway. Knockdown of FOXM1 by RNA interference inhibited cell proliferation by arresting cells in G2/M and reduced cell invasion and motility. This phenotype was associated with decreased expression of FOXM1 target genes, like cyclin B1 (CCNB1), polo-like kinase 1 (PLK1), Aurora B (AURKB), S-phase kinase-associated protein 2 (SKP2), and plasminogen activator, urokinase: uPA (PLAU). Pharmacological inhibition of FOXM1 in an orthotopic mouse model of ATC reduced tumor burden and metastasization. All together, these findings suggest that FOXM1 represents an important player in thyroid cancer progression to the anaplastic phenotype and a potential therapeutic target for this fatal cancer.