Thyroid Tumor Cell Lines Research Articles

Estrogen-related receptor alpha modulates lactate dehydrogenase activity in thyroid tumors.

Metabolic modifications of tumor cells are hallmarks of cancer. They exhibit an altered metabolism that allows them to sustain higher proliferation rates in hostile environment outside the cell. In thyroid tumors, the expression of the estrogen-related receptor α (ERRα), a major factor of metabolic adaptation, is closely related to the oxidative metabolism and the proliferative status of the cells. To elucidate the role played by ERRα in the glycolytic adaptation of tumor cells, we focused on the regulation of lactate dehydrogenases A and B (LDHA, LDHB) and the LDHA/LDHB ratio. Our study included tissue samples from 10 classical and 10 oncocytic variants of follicular thyroid tumors and 10 normal thyroid tissues, as well as samples from three human thyroid tumor cell lines: FTC-133, XTC.UC1 and RO82W-1. We identified multiple cis-acting promoter elements for ERRα, in both the LDHA and LDHB genes. The interaction between ERRα and LDH promoters was confirmed by chromatin immunoprecipitation assays and in vitro analysis for LDHB. Using knock-in and knock-out cellular models, we found an inverse correlation between ERRα expression and LDH activity. This suggests that thyroid tumor cells may reprogram their metabolic pathways through the up-regulation of ERRα by a process distinct from that proposed by the recently revisited Warburg hypothesis.

An antibody-like peptide that recognizes malignancy among thyroid nodules

There is an urgent need for biomarkers to identify malignant thyroid nodules from indeterminate follicular lesions. We have used a subtractive proteomic strategy to identify novel biomarkers by selecting ligands to goiter tissue from a 12-mer random peptide phage-displayed library using the BRASIL method (Biopanning and Rapid Analysis of Selective Interactive Ligands). After three rounds of selection, two highly reactive clones to the papillary thyroid tumor cell line NPA were further evaluated, and their specific binding to tumor proteins was confirmed using phage-ELISA. The antibody-like peptide CaT12 was tumor-specific, which was further tested by immunohistochemistry against TMAs (tissue microarrays) comprised of 775 human benign and malignant tissues, including 232 thyroid nodular lesions: 15 normal thyroid tissues, 53 nodular goiters (NG), 54 follicular adenomas (FA); 69 papillary thyroid carcinomas (PTC); and 41 follicular carcinomas (FC). CaT12 was able to identify PTC among thyroid nodular lesions with 91.2% sensitivity and 85.1% specificity, despite its non-specificity for thyroid tissues. Additionally, the CaT12 peptide helped characterize follicular lesions distinguishing the follicular variant of PTC (FVPTC) from FA with 91.9% accuracy; FVPTC from NG with 83.1% accuracy; FVPTC from the classic PTC with 57.7% accuracy; and FVPTC from FC with 88.7% accuracy. In conclusion, our strategy to select differentially expressed ligands to thyroid tissue was highly effective and resulted in a useful antibody-like biomarker that recognizes malignancy among thyroid nodules and may help distinguish follicular patterned lesions.

A Human Thyroid Cancer Cell Line, DH-14-3, Newly Established from Poorly Differentiated Thyroid Carcinoma

Poorly differentiated thyroid carcinoma (PDTC) is a newly recognized histological type of malignant thyroid tumor, accounting for about 2 - 13% of all thyroid carcinomas. PDTC is considered as a morphologically and biologically intermediate stage between well-differentiated thyroid carcinoma and anaplastic thyroid carcinoma. PDTC preferentially manifests bone metastases. We here established a cell line from a resected tumor specimen from a 70-year-old male patient with PDTC who presented with multiple bone metastases. This new thyroid tumor cell line was designated as DH-14-3 and was subsequently grown in culture for several years. DH-14-3 cells express thyroglobulin in the cytoplasm and thyroid transcription factor-1 in the nuclei, both proteins of which are specific markers for the thyroid gland. Importantly, triiodothyronine (T3) was detected in the cultured medium of DH-14-3 cells, in which, however, thyroxine (T4) was undetectable. Moreover, DH-14-3 cells secreted interleukin-8, transforming growth factor-β1, vascular endothelial growth factor, matrix metalloproteinase-1 and parathyroid hormone-related protein, all of which may be responsible for the aggressiveness or bone metastasis of PDTC. Thus, the production of these proteins may reflect the metastatic potential of this cell line. DH-14-3 cells also express CXC chemokine receptor-4 and epidermal growth factor receptor, and carry a missense mutation in the p53 tumor suppressor gene. In fact, transplantation of DH-14-3 cells into the back of nude mice resulted in the formation of tumors, thereby confirming the capability of tumorigenesis. DH-14-3 cells may be useful for investigating the biological features of PDTC and will contribute to the therapeutic study of thyroid cancer.

Overexpression of pro-inflammatory genes and down-regulation of SOCS-1 in human PTC and in hypoxic BCPAP cells

Hypoxia-inducible factor-1α (HIF-1α) is frequently overexpressed and activated in many cancer types. However, its regulation and function in thyroid carcinomas are only partially known. Aim of our study was to demonstrate that adaptation to the hypoxic micro-environment by human papillary thyroid carcinoma (PTC) cells, in the absence of leukocyte infiltrate, induces a “molecular inflammation” process characterized by the expression of a large set of genes normally involved in inflammation. To address this, tumor, peritumor or normal host tissue from eleven human PTC surgical samples, were separated by laser capture microdissection (LCMD) and studied by real-time quantitative PCR and Western blot. In such condition, we observed an increased expression and activation of HIF-1α, NF-kB and pro-inflammatory genes only in tumor tissues. Importantly, an anti-inflammatory gene such as SOCS-1 was markedly down-regulated in tumor tissue compared to surrounding normal host tissue. Similar results were found in fine-needle aspiration biopsy (FNAB)-derived specimens from PTC and in hypoxic human papillary thyroid tumor cell line, BCPAP. Moreover, we also detected an elevated expression of metalloproteinase-9 (MMP9) both in solid tumor and in hypoxic-treated BCPAP cells. Our findings reveal that, in human PTC tumor, hypoxic conditions are accompanied by up-regulation of pro-inflammatory genes, down-regulation of anti-inflammatory genes and increased expression of MMP9. We propose that a better understanding of the pro- and anti-inflammatory pathways involved in the “molecular inflammation” process even in the absence of leukocyte, may help to clarify progression toward malignancy and may prove useful for new anti-tumor strategy.

Activation of the Sonic Hedgehog pathway in thyroid neoplasms and its potential role in tumor cell proliferation

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.

Histone deacetylase inhibitors control the transcription and alternative splicing of prohibitin in thyroid tumor cells

Prohibitin (PHB) is a ubiquitous protein with a number of different molecular functions. PHB is involved in tumorigenesis by exerting either a permissive or blocking action on tumor growth, depending on the cell context. In the present study, we investigated the effects of the histone deacetylase inhibitors (HDACis), trichostatin A (TSA) and sodium butyrate (NaB), on PHB expression in the thyroid tumor cell lines, TPC-1 and FRO. Both TSA and NaB increased PHB mRNA levels. Transfection experiments showed that the overexpression of HDAC1 or 2, but not 3, inhibited PHB promoter activity. The effects of TSA and NaB on the two major PHB mRNA splicing isoforms, were also investigated. Both TSA and NaB decreased the mRNA levels of the shorter isoform, but increased those of the longer isoform. Only the latter isoform contains a 3'UTR, which has been reported to exert a growth suppressive action. Thus, our data demonstrate that HDACis control both PHB transcription and alternative splicing. The effect of HDACis on PHB alternative splicing was not due to the modification of the expression of the ASF/SF2 splicing factor.

HMGA2 gene expression analysis performed on cytologic smears to distinguish benign from malignant thyroid nodules

Up to 80% of thyroid nodules with an indeterminate diagnosis on fine-needle aspiration (FNA) (eg, "suspicious for follicular neoplasm") prove to be benign at the time of surgical resection. Ancillary tests in current use are limited in their ability to improve the preoperative detection of malignant follicular thyroid nodules. Studies using paraffin-embedded tissue have indicated that high mobility group AT-hook 2 (HMGA2) overexpression is present in a high percentage of malignant thyroid neoplasms but not in benign thyroid neoplasms. In the current study, the ability of HMGA2 overexpression analysis to preoperatively distinguish benign from malignant thyroid nodules by reverse transcriptase-polymerase chain reaction (RT-PCR) on suspicious cytologic smears was evaluated. Patients who underwent thyroid FNA and subsequent thyroid resection from 2001 through 2007 were identified. A subset of these patients who had a cytologic diagnosis of "suspicious" underwent HMGA2 expression analysis. HMGA2 expression was detected on cells scraped from cytologic smears with 1-step, real-time quantitative RT-PCR. Of the 125 cases identified, RNA extraction and HMGA2 analysis were successful in 115 cases. With an HMGA2 overexpression change of 5.9-fold or greater compared with a thyroid tumor cell line as a positive cutoff, the test was found to have the following overall performance for detecting malignant nodules: sensitivity of 71%, specificity of 97%, positive predictive value of 94%, and negative predictive value of 84%. HMGA2 overexpression was found to have low sensitivity for detecting Hurthle cell carcinoma (33%). HMGA2 mRNA expression analysis can be performed on cytologic smears and demonstrates a high specificity and positive predictive value and relatively high sensitivity and negative predictive value for detecting malignancy in "suspicious" thyroid aspirate specimens.

Nucleophosmin is overexpressed in thyroid tumors

Nucleophosmin (NPM) is a protein that contributes to several cell functions. Depending on the context, it can act as an oncogene or tumor suppressor. No data are available on NPM expression in thyroid cells. In this work, we analyzed both NPM mRNA and protein levels in a series of human thyroid tumor tissues and cell lines. By using immunohistochemistry, NPM overexpression was detected in papillary, follicular, undifferentiated thyroid cancer, and also in follicular benign adenomas, indicating it as an early event during thyroid tumorigenesis. In contrast, various levels of NPM mRNA levels as detected by quantitative RT-PCR were observed in tumor tissues, suggesting a dissociation between protein and transcript expression. The same behavior was observed in the normal thyroid FRTL5 cell lines. In these cells, a positive correlation between NPM protein levels, but not mRNA, and proliferation state was detected. By using thyroid tumor cell lines, we demonstrated that such a post-mRNA regulation may depend on NPM binding to p-Akt, whose levels were found to be increased in the tumor cells, in parallel with reduction of PTEN. In conclusion, our present data demonstrate for the first time that nucleophosmin is overexpressed in thyroid tumors, as an early event of thyroid tumorigenesis. It seems as a result of a dysregulation occurring at protein and not transcriptional level related to an increase of p-Akt levels of transformed thyrocytes.

Anti-tumour activity of CS-7017, a selective peroxisome proliferator-activated receptor gamma agonist of thiazolidinedione class, in human tumour xenografts and a syngeneic tumour implant model

The anti-tumour activity of the novel thiazolidinedione class peroxisome proliferator-activated receptor gamma (PPARγ) agonist CS-7017 was investigated. CS-7017 activated PPARγ-mediated luciferase expression with an EC50 of 0.20nM. In addition, CS-7017 was shown to be highly selective for PPARγ amongst other PPAR subfamilies. CS-7017 inhibited the proliferation of the human anaplastic thyroid tumour cell line DRO and the pancreatic tumour cell line AsPC-1 in vitro at concentrations as low as 10nM. In xenograft studies, CS-7017 inhibited the growth of the human colorectal tumour cell line HT-29 in nude mice as well as DRO in nude rats in a dose-dependent manner. At the same dose, an increase in the levels of adiponectin, a surrogate marker for PPARγ activation, was also observed. CS-7017 prolonged the survival of mice inoculated with murine colorectal tumour Colon 38 with marginal tumour growth inhibition. These preclinical results support the potential utility of CS-7017 in a clinical setting.

Expression analysis of facilitative glucose transporters (GLUTs) in human thyroid carcinoma cell lines and primary tumors

Fluorine-18-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET) is based on cell capability to take-up glucose. While a significantly higher expression of the glucose transporter GLUT1 has been reported in thyroid tumors only few data are available on the expression of other GLUT isoforms. We studied several GLUT isoforms expression in thyroid tumor cell lines deriving from anaplastic (ARO, FRO), papillary (NPA), follicular (WRO) and medullary (TT) human thyroid carcinoma. GLUT1 and GLUT3 were also studied in 157 human thyroid malignant and benign tissues. Quantitative Real-time RT-PCR analysis revealed that GLUT1 mRNA levels were higher in less-differentiated cells (ARO, FRO) while GLUT3 mRNA levels were prevalent in well-differentiated cells (NPA, WRO). Accordingly, Western blot showed high expression and correct membrane targeting of GLUT1 protein in ARO and FRO and of GLUT3 protein in NPA and WRO. All cell lines were able to take-up different rates of (3)H-deoxy-glucose. The analysis of GLUT1 and GLUT3 mRNA expression in human thyroid tissues showed the prevalence of GLUT1, but not of GLUT3, in malignant with respect to normal tissues. Finally, both GLUT1 and GLUT3 showed a slightly higher expression in anaplastic than in well-differentiated tumors. In conclusion, we showed that GLUT1 and GLUT3 were the most important glucose transporters in the thyroid tumoral cells. In particular GLUT1 was the most prevalent in less-differentiated cells (ARO and FRO) while GLUT3 was the most prevalent in well-differentiated cells (NPA and WRO). A similar pattern of expression was found for GLUT1 but not for GLUT3 in human thyroid tumors.

High periostin expression correlates with aggressiveness in papillary thyroid carcinomas

Periostin is a mesenchyme-specific gene product, which acts as an adhesion molecule during bone formation and supports osteoblastic cell line attachment and spreading. However, periostin expression is activated in a large variety of epithelial human tumors and correlates with their aggressiveness. Knowledge of expression of periostin in thyroid tumors is still scanty. The aim of the present work was to investigate periostin expression in differentiated neoplasms of the thyroid and to correlate it with several clinical and molecular features of these tumors. Periostin expression was evaluated by quantitative PCR and immunohistochemistry in normal thyroid tissues, papillary thyroid carcinomas (PTCs), follicular thyroid carcinomas (FTCs), and follicular adenomas (FAs). Periostin mRNA levels were also evaluated in several thyroid tumor cell lines. PTCs show mean periostin mRNA levels significantly higher than corresponding normal tissues. In five PTCs, periostin mRNA values were at least 30-fold higher than corresponding normal tissues. Conversely, mean periostin mRNA levels of FTCs and FAs were similar to those of normal tissues. Consistent with mRNA studies, periostin was detectable by immunohistochemistry in cancerous epithelial cells only in several cases of PTCs but not in normal tissue, FTCs, and FAs. In PTCs, periostin mRNA levels positively correlate with extrathyroidal invasion, distant metastasis, and higher grade staging. A negative correlation between periostin and expression of some markers of the thyroid-differentiated phenotype (thyroglobulin, thyrotropin receptor) was also present in the PTCs. These results indicate that an increase in periostin gene expression is present in several PTCs, in which it appears as a marker of aggressiveness. Experiments in thyroid tumor cell lines indicate that high levels of periostin mRNA are due, at least in part, to the increase in periostin promoter activity.

Lack of Mutations in the Thyroid Hormone Receptor (TR) α and β Genes but Frequent Hypermethylation of the TRβ Gene in Differentiated Thyroid Tumors

It remains inconclusive whether mutations in thyroid hormone receptor (TR) genes naturally occur in thyroid cancer and whether these genes could be suppressors of this cancer. Our objectives were to examine further mutations of TRalpha and TRbeta genes in thyroid cancer and also to examine their methylation as an epigenetic silencing mechanism in thyroid cancer. Instead of using a cDNA sequencing approach used in previous studies, we used genomic DNA to sequence directly the coding regions of the TRalpha and TRbeta genes to search mutations in various differentiated thyroid tumors and used methylation-specific PCR to analyze promoter methylation of these genes. Allelic zygosity status at TRbeta was also analyzed. We found no TRalpha gene mutation in 17 papillary thyroid cancers (PTCs) and 11 follicular thyroid cancers (FTCs), and no TRbeta gene mutation in 16 PTCs and 12 FTCs. We also found no methylation of the TRalpha gene in 33 PTCs, 31 FTCs, 20 follicular thyroid adenomas (FTAs), and 10 thyroid tumor cell lines. In contrast, we found hypermethylation of the TRbeta gene in 10 of 29 (34%) PTCs, 22 of 27 (81%) FTCs, five of 20 (25%) follicular thyroid adenomas, and three of 10 (30%) thyroid tumor cell lines, with the highest prevalence in FTC. We additionally examined loss of heterozygosity at TRbeta and found it in three of nine (33%) PTCs and three of nine (33%) FTCs. Mutation is not common in TR genes, whereas hypermethylation of the TRbeta gene as an alternative gene silencing mechanism is highly prevalent in thyroid cancer, particularly FTC, consistent with a possible tumor suppressor role of this gene for FTC.

Human Thyroid Tumor Cell Lines Derived from Different Tumor Types Present a Common Dedifferentiated Phenotype

Cell lines are crucial to elucidate mechanisms of tumorigenesis and serve as tools for cancer treatment screenings. Therefore, careful validation of whether these models have conserved properties of in vivo tumors is highly important. Thyrocyte-derived tumors are very interesting for cancer biology studies because from one cell type, at least five histologically characterized different benign and malignant tumor types can arise. To investigate whether thyroid tumor-derived cell lines are representative in vitro models, characteristics of eight of those cell lines were investigated with microarrays, differentiation markers, and karyotyping. Our results indicate that these cell lines derived from differentiated and undifferentiated tumor types have evolved in vitro into similar phenotypes with gene expression profiles the closest to in vivo undifferentiated tumors. Accordingly, the absence of expression of most thyrocyte-specific genes, the nonresponsiveness to thyrotropin, as well as their large number of chromosomal abnormalities, suggest that these cell lines have acquired characteristics of fully dedifferentiated cells. They represent the outcome of an adaptation and evolution in vitro, which questions the reliability of these cell lines as models for differentiated tumors. However, they may represent useful models for undifferentiated cancers, and by their comparison with differentiated cells, can help to define the genes involved in the differentiation/dedifferentiation process. The use of any cell line as a model for a cancer therefore requires prior careful and thorough validation for the investigated property.

In vitro characterization of the thyroidal uptake of O-(2-[18F]fluoroethyl)-l-tyrosine

Positron emission tomography (PET) using O-(2-[(18)F]fluoroethyl)-l-tyrosine (FET) has been successfully employed in the diagnostic workup of brain tumors. Knowledge on the mechanisms of the uptake of radiolabeled amino acids into thyroidal tissues and well-differentiated thyroid carcinomas is limited. We therefore studied several factors potentially governing the uptake of FET in the rat thyroid cell line FRTL-5 in comparison with thyroid tumor cell lines of human origin. FET uptake was determined in thyroid-stimulating hormone (TSH)-stimulated and TSH-deprived FRTL-5 cells, as well as in the cell lines U-138 MG (human glioblastoma), Onco DG-1 (human papillary thyroid carcinoma) and ML-1 (human follicular thyroid carcinoma). The TSH responsiveness of cells was measured by the incubation of TSH-treated and untreated control cells with 2-[(18)F]fluoro-2-deoxyglucose (FDG). All cellular tracer uptake values were related to total protein mass and expressed as percentage per milligram. For countertransport studies, FRTL-5 cells were exposed to 10-300 microM tyrosine methyl ester. TSH-stimulated and TSH-deprived FRTL-5 cells were incubated with 100 kBq/ml FET for 20 min. 2-Aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH), alpha-(methylamino)-isobutyric acid, L-serine and tryptophan were used as competitive inhibitors of FET uptake. All inhibition experiments were repeated with the human thyroid carcinoma cell lines to obtain comparative FET uptake values. The FET uptake was 155+/-30%/mg in FRTL-5 cells (n=6), 108+/-14%/mg in U-138 MG cells (n=6), 194+/-60%/mg in ML-1 cells (n=9) and 64+/-23%/mg in Onco DG-1 cells (n=6) under identical incubation conditions. Preloading with tyrosine methyl ester increased cellular FET uptake dose dependently in FRTL-5 cells (165+/-25%, n=6). While TSH increased the uptake of FDG in FRTL-5 cells by sixfold, there was no TSH effect on FET accumulation. FET uptake by TSH-treated FRTL-5 cells was sodium independent and significantly inhibited by BCH (91.4+/-3.0%, n=9), tryptophan (94.8+/-1.6%, n=8) and serine (83.2+/-10.8%, n=12). TSH-starved FRTL-5 cells had a sodium-dependent component with a similar inhibition pattern. Onco DG-1 mainly confirmed the inhibition pattern of FET uptake in FRTL-5 cells, reflecting System-L-mediated FET uptake that was blocked by BCH and serine (72-85%, n=9). ML-1 cells revealed a pronounced sodium-dependent FET uptake that was inhibited by tryptophan (70+/-10%, n=9, P<.05) in the presence and in the absence of sodium, suggesting a contribution of alternative amino acid carriers. FET uptake by FRTL-5 cells is not TSH dependent. FET uptake by FRTL-5 cells seems to be mainly mediated by a carrier exhibiting the characteristics of the System L amino acid transporter. FET uptake in thyroid cells and thyroid carcinoma cells was in the same range as that in a glioblastoma cell line. This encourages further research efforts towards the clinical evaluation of FET for the diagnostic workup of well-differentiated thyroid carcinomas.

RIZ1 is epigenetically inactivated by promoter hypermethylation in thyroid carcinoma

Allelotype studies have suggested that chromosome 1p is frequently lost in thyroid cancers, thus suggesting that there is an important tumor suppressor at this location. RIZ1 (PRDM2), located on 1p36, is a recently described tumor suppressor gene and is a member of the protein methyltransferase superfamily. RIZ1 expression is lost in a variety of tumors, primarily by means of epigenetic mechanisms that involve promoter hypermethylation. RIZ1 expression was examined in a panel of thyroid tumor cell lines and primary thyroid tissues (14 normal, 19 benign, and 31 cancerous) by using real-time polymerase chain reaction (PCR). Methylation status of the RIZ1 promoter was studied using bisulfite sequencing and methylation-specific PCR. The authors demonstrated that RIZ1 expression is lost in thyroid tumor cell lines and is also significantly reduced in thyroid carcinomas, when compared with normal thyroid tissues (P < .0001) and benign tumors (P = .0003). The current study results also showed that loss of RIZ1 is mediated by aberrant cytosine methylation of the RIZ1 promoter. One hundred percent of carcinomas were methylated, compared with 33% of normal thyroid tissues (P = .001). RIZ1 mRNA expression was significantly higher (P = .02) in unmethylated (1.22 +/- 1.2, mean +/- standard deviation [SD]), compared with methylated tissues (0.37 +/- 0.42, mean +/- SD). Last, treatment with a DNA methyltransferase inhibitor led to reactivation of RIZ1 expression in cell lines that had negligible RIZ1 expression at baseline. The current study suggested an important role for RIZ1 expression in thyroid tumorigenesis and identified a potential novel therapeutic target for tumors unresponsive to other therapies.

Expression and function of the chemokine receptor CCR7 in thyroid carcinomas

The chemokine receptor CCR7 plays a critical role in lymphocyte and dendritic cell trafficking into and within lymph nodes, the preferential metastatic site for papillary (PTC) and medullary (MTC) thyroid carcinomas. In order to determine a possible role for CCR7 in mediating the metastatic behaviour of thyroid carcinomas, we analysed its expression in normal and tumoral thyroid tissues of different histotypes and studied the in vitro effects of its activation by the CCR7 ligand, CCL21. Using real-time quantitative-PCR, we observed that CCR7 expression was higher in PTCs and MTCs than in follicular and poorly differentiated thyroid carcinomas. CCR7 expression was ninefold higher in classic compared with follicular variants of PTCs, and its expression in MTCs was significantly correlated with lymph node metastases. Immunohistochemical staining for CCR7 showed protein expression in neoplastic thyroid cells, with higher intensity in PTCs, MTCs and their lymph node metastases (LNMs). We further showed that CCL21 stimulation of a CCR7-expressing thyroid tumour cell line (TPC-1) promotes cell proliferation and migration, and the chemotactic effect of CCL21 in these cells involves actin polymerization, increased beta1-integrin expression and increased matrix metalloproteinase secretion. Taken together, our results demonstrate that CCR7 activation on thyroid carcinoma cells by CCL21 - a chemokine abundantly expressed in lymph nodes - favours tissue invasion and cell proliferation, and therefore may promote thyroid carcinoma growth and LNM.

Clinicopathological Significance of Major Histocompatibility Complex Class I-Related Chain A and B Expression in Thyroid Cancer

Major histocompatibility complex class I-related chains A and B (MICA/B) are two stress-inducible ligands for the immunoreceptor NKG2D that is expressed on cytotoxic T cells and natural killer (NK) cells. It is not known whether MICA/B expression is up-regulated in thyroid cancer as a result of oncogene activation. The objective of the investigation was to study MICA/B expression and regulation in thyroid cancer and its role in mediating the cytotoxicity of NK cells. MICA/B expression in thyroid cancer was analyzed by immunohistochemical staining. Cell surface MICA/B levels in thyroid tumor cell lines and fresh tumor cells were analyzed by flow cytometric analysis. The susceptibility of thyroid tumor cells to NK cell killing was tested by using (51)Cr release assay. MICA/B was expressed at moderate or high levels in 18 of 39 papillary thyroid carcinomas and four of eight anaplastic thyroid carcinomas. MICA/B expression was confirmed by flow cytometric analysis in three fresh thyroid neoplasms. MICA/B expression was detected in eight of 10 thyroid tumor cell lines and correlated with their sensitivity to killing by the NKG2/D-positive NK-92 cells. Blocking of NKG2D and MICA/B interaction by specific antibodies partially led to the inhibition of NK-92 cell-mediated cytotoxicity. The MAPK inhibitors were able to block MICA/B expression in MRO87 and HeLa cells. Transient transfection of mutant BRAF and RAS oncogenes led to increased MICA/B expression in 293 cells and WRO82 cells. MICA/B expression is up-regulated in thyroid cancer, probably due to the activation of the MAPK pathway. MICA/B in thyroid cancer plays an important role in NK-92 cell-mediated cytotoxicity.

Mitochondrial Activities of a Cell Line Derived from Thyroid Hürthle Cell Tumors

We conducted a study on the functional characteristics of mitochondria in an oxyphilic thyroid tumor cell line, which may provide useful clues about Hürthle cell tumors carcinogenesis. The functional study on thyroid tumors with cell oxyphilia (Hürthle cell tumors), characterized by mitochondrial hyperplasia, was carried out in XTC.UC1, and B-CPAP, an oxyphilic and nonoxyphilic thyroid tumor cell line, respectively. XTC.UC1 cell line showed higher activity of mitochondrial respiratory complexes I and II and decreased activity of complex III. The increased activity of complex I was not matched by increased expression of complex I subunits. The XTC.UC1 cells relied mostly on oxidative phosphorylation for energy conservation, although their mitochondrial energetic function was less efficient when related to mitochondrial content of the cells. Finally, the oxyphilic cell line produced significantly higher amounts of reactive oxygen species (ROS) in comparison with B-CPAP cell line. The involvement of ROS in mitochondrial biogenesis and proliferation as well as in carcinogenesis and apoptosis indicate that differences in activity of respiratory chain components and their unbalance may be responsible for development of morphological and functional changes observed in thyroid tumors with cell oxyphilia.

Expression of Matrix Metalloproteinases and Their Specific Inhibitors in Normal and Different Human Thyroid Tumor Cell Lines

Expression of Matrix Metalloproteinases and Their Specific Inhibitors in Normal and Different Human Thyroid Tumor Cell Lines

Identification of an Aberrantly Spliced Form of HDMX in Human Tumors: A New Mechanism for HDM2 Stabilization

The HDMX protein is closely related to HDM2 with which it shares different structural domains, particularly the p53 binding domain and the ring finger domain, where the two HDM proteins interact. Several oncogenic forms derived from splicing of HDM2 have been described in cancer. This work aimed at investigating whether analogous forms of HDMX exist in human tumors. Here, we report the characterization of an aberrantly spliced form of HDMX, HDMX211, isolated from the thyroid tumor cell line, ARO. HDMX211 binds and stabilizes the HDM2 protein. Although it lacks the p53 binding domain, HDMX211 also stabilizes p53 by counteracting its degradation by HDM2. However, the resulting p53 is transcriptionally inactive and increasingly associated to its inhibitor HDM2. Expression of HDMX211 strongly enhances the colony-forming ability of human cells in the presence or absence of wild-type p53. Conversely, depletion of HDMX211 by small interfering RNA significantly reduces the growth of ARO cells and increases their sensitivity to chemotherapy. Screening of lung cancer biopsies shows the presence of HDMX211 in samples that overexpress HDM2 protein, supporting a pathologic role for this new protein. This is the first evidence of a variant form of HDMX that has oncogenic potential independently of p53. HDMX211 reveals a new mechanism for overexpression of the oncoprotein HDM2. Most interestingly, it outlines a possible molecular explanation for a yet unclarified tumor phenotype, characterized by simultaneous overexpression of HDM2 and wild-type p53.