epidermal-growth-factor and Thyroid-Neoplasms

Medullary thyroid carcinoma (MTC) is a rare endocrine malignancy accounting for a significant percentage of thyroid cancer-related fatal events. Traditional treatment modalities used in the other types of thyroid carcinomas have been proved largely ineffective in advanced MTC. Better understanding of the molecular pathways implicated in the pathogenesis of MTC has led to the development of new drugs, which are implicated in the disruption of these molecular cascades.. This review provides the latest information regarding vandetanib , a new tyrosine kinase inhibitor mainly in the treatment of MTC. A collection of available data was conducted using the PubMed database as well as the ClinicalTrials.gov website, searching for vandetanib and thyroid cancer.. Vandetanib targets multiple cell-signaling pathways involved in the molecular pathogenesis of thyroid cancer, namely vascular endothelial growth factor receptor-2, epidermal growth factor receptor and rearranged during transfection receptor. It is an effective approach in treating advanced MTC. However, treatment toxicity issues, as well as individual patient parameters, including disease burden and progression, should be taken into consideration before initiating vandetanib treatment.

Topics: Animals; Carcinoma, Neuroendocrine; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Disease Models, Animal; Drug Evaluation, Preclinical; Epidermal Growth Factor; Humans; Piperidines; Protein Kinase Inhibitors; Quinazolines; Randomized Controlled Trials as Topic; Thyroid Neoplasms; Transfection; Treatment Outcome; Vascular Endothelial Growth Factor Receptor-2

Thrombospondin-1 (TSP-1) is a member of a family of five structurally related extracellular glycoproteins that plays a major role in cell-matrix and cell-cell interactions. Due to its multifunctional nature and its ability to bind to a variety of cell surface receptors and matrix proteins, TSP-1 has been identified as a potential regulator of angiogenesis and tumor progression. In this review, we summarize recent results that we obtained in our laboratory dealing with the regulation of thombospondin-1 expression by epidermal growth factor and hepatocyte growth factor. Our results show that TSP-1 can have opposite effects on cell invasion depending upon the type of differentiated thyroid carcinoma studied.

Topics: Carcinoma, Papillary; Carcinoma, Papillary, Follicular; Down-Regulation; Epidermal Growth Factor; Extracellular Matrix; Hepatocyte Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Thrombospondin 1; Thyroid Neoplasms; Up-Regulation

Iodine is a trace element that is essential for the synthesis of thyroid hormone. Both chronic iodine deficiency and iodine excess have been associated with hypertrophy and hyperplasia of follicular cells, attributed to excessive secretion of TSH. This may be associated to thyroid cancer risk, particularly in women. Experimental studies have documented thyroid cancer induction by elevation of endogenous TSH, although in a small number of animals. Iodine deficiency associated with carcinogenic agents and chemical mutagens will result in a higher incidence of thyroid malignancy. Inadequate low iodine intake will result in increased TSH stimulation, increased thyroid cell responsiveness to TSH, increased thyroid cell EGF-induced proliferation, decreased TGFbeta 1 production and increased angiogenesis, all phenomena related to promotion of tumor growth. Epidemiological studies associating iodine intake and thyroid cancer led to controversial and conflicting results. There is no doubt that introduction of universal iodine prophylaxis in population previously in chronic iodine-deficiency leads to a changing pattern of more prevalent papillary thyroid cancer and declining of follicular thyroid cancer. Also anaplastic thyroid cancer is practically not seen after years of iodine supplementation. Iodine excess has also been indicated as a possible nutritional factor in the prevalence of differentiated thyroid cancer in Iceland, Hawaii and, more recently, in China.. available evidence from animal experiments, epidemiological studies and iodine prophylaxis has demonstrated a shift towards a rise in papillary carcinoma, but no clear relationship between overall thyroid cancer incidence and iodine intake.

Topics: Adenocarcinoma, Follicular; Adenocarcinoma, Papillary; Animals; Argentina; Diet; Disease Models, Animal; Epidemiologic Studies; Epidermal Growth Factor; Female; Hawaii; Humans; Iceland; Iodine; Italy; Male; Thyroid Gland; Thyroid Neoplasms; Thyrotropin

Topics: Animals; Cell Division; Epidermal Growth Factor; Humans; Receptors, Thyrotropin; Thyroid Diseases; Thyroid Gland; Thyroid Neoplasms; Thyrotropin

Growth of thyroid cancer cells is stimulated by various growth factors via signal transduction pathways. TSH, EGF, IGF, and TGF-alpha stimulate and TGF-beta inhibits thyroid cell growth. TSH stimulates thyroid cells via both the adenylate cyclase-PKA and the PLC-PKC-Ca signal transduction pathways. TSH-r, ras, gsp, ret, trk, and myc are oncogenes that are activated in some thyroid neoplasms. P53 and RB are tumor suppressor genes that are inactivated in some thyroid cancers.

Topics: Epidermal Growth Factor; Humans; Oncogenes; Signal Transduction; Somatomedins; Thyroid Gland; Thyroid Neoplasms; Thyrotropin; Transforming Growth Factor alpha; Transforming Growth Factor beta

TSH activates the adenylate cyclase and the phosphoinositide turnover-protein kinase C-calcium systems in thyroid cells and appears to have an important but variable role in controlling the growth of both normal and neoplastic thyroid tissues. Species differences, experimental conditions, and tissue or tumor cell heterogeneity may account for this variability. Although TSH seems to be an important physiologic growth factor, it is neither the exclusive growth factor for the thyroid gland nor absolutely necessary for the effect of other thyroid growth factors. TSH may work in concert with other growth factors such as EGF. Some growth factors influence thyroid growth through TSH, whereas others do not.

Topics: Adenylyl Cyclases; Epidermal Growth Factor; Humans; Phosphatidylinositols; Protein Kinase C; Receptors, Thyrotropin; Thyroid Gland; Thyroid Neoplasms; Thyrotropin

Cellular retinoic acid binding protein 1 (CRABP1) participates in the regulation of retinoid signaling. Previous studies showed conflicting results regarding the role of CRABP1 in tumor biology, including protumorigenic and tumor-suppressive effects in different types of cancer. Our bioinformatics analyses suggested that CRABP1 expression was downregulated in thyroid cancer. Ectopic expression of CRABP1 in thyroid cancer cells suppressed migratory and invasive activity without affecting cell growth or cell cycle distribution. In transformed normal thyroid follicular epithelial cells, silencing of CRABP1 expression increased invasiveness. Additionally, CRABP1 overexpression was associated with downregulation of the mesenchymal phenotype. Kinase phosphorylation profiling indicated that CRABP1 overexpression was accompanied by a decrease in phosphorylation of epidermal growth factor (EGF) receptor and downstream phosphorylation of Akt, STAT3, and FAK, which were reversed by exogenous EGF treatment. Immunohistochemical analysis of our tissue microarrays revealed an inverse association between CRABP1 expression and disease stage of differentiated thyroid cancer. Taken together, our results suggest that CRABP1 expression is aberrantly lost in thyroid cancer, and this downregulation promotes the epithelial-mesenchymal transition at least partly through modulating EGF receptor signaling.

Topics: Cell Line, Tumor; Down-Regulation; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Humans; Receptors, Retinoic Acid; Thyroid Neoplasms

Epidermal growth factor (EGF) has various important physiological functions, which it exerts by binding to the epidermal growth factor receptor (EGFR). Reports show that EGF expression is strongly correlated with the occurrence and development of many types of tumour. To date, however, the relationship between EGF/EGFR and the occurrence and development of thyroid carcinoma remains unclear.. In the current study, we investigated this phenomenon using human anaplastic thyroid carcinoma cell lines (SUN-80).. The results indicated that EGF triggered the EGFR-mediated intracellular signalling pathway, including signal transducers and activators of transcription 1/3/5 (STAT1/3/5) and protein kinase B (AKT) in a time- and dose-dependent manner. In addition, results from EGF-induced EGFR internalization and co-localization analyses showed that clathrin, Rab5/7, and EEA1 play critical roles in the intracellular trafficking of EGF/EGFR. Interestingly, EGF triggered EGFR translocation into the nucleus, while nuclear-localized EGFR affected cell cycle distribution, thereby significantly promoting the ration of S phase. Overall, these findings indicated that nuclear EGFR exerts biological activity and physiological functions, including changing cell cycle, which in turn promotes proliferation and migration of SUN-80 cells.. These findings lay a foundation for further explorations seeking to understand the biological effects of the EGF/EGFR system on the occurrence and development of thyroid cancer.

Topics: Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Humans; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms

Hypoxia-induced angiogenesis functions importantly in anaplastic thyroid cancer (ATC) progression. However, the therapeutic potential of broad-spectrum anti-angiogenic agent remains undefined. Anlotinib conventionally targets VEGFR, FGFR and PDGFR. Here, a novel role of anlotinib on ATC angiogenesis was illustrated.. Molecular expressions were established via tissue microarray. Multiple assays (tubule formation, 3D sprouting and chicken chorioallantoic membrane model) were used for angiogenic evaluation. Panels of molecular screening were achieved by antibody and PCR arrays. The loop binding motif of EGFR for homology modelling was prepared using Maestro.. Anlotinib could dose- and time-dependently inhibit cell viability under normoxia and hypoxia and could repress hypoxia-activated angiogenesis more efficiently in vitro and in vivo. CXCL11 and phospho-EGFR were hypoxia-upregulated with a positive correlation. The cancer-endothelium crosstalk could be mediated by the positive CXCL11-EGF-EGFR feedback loop, which could be blocked by anlotinib directly targeting EGFR via a dual mechanism by simultaneous inhibitory effects on cancer and endothelial cells. The AKT-mTOR pathway was involved in this regulatory network.. The newly identified CXCL11-EGF-EGFR signalling provided mechanistic insight into the interaction between cancer and endothelial cells under hypoxia, and EGFR was a novel target. Anlotinib may be the encouraging therapeutic candidate in ATC.

Topics: Animals; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Chemokine CXCL11; Epidermal Growth Factor; ErbB Receptors; Feedback, Physiological; Female; Gene Expression Regulation, Neoplastic; Humans; Indoles; Mice; Protein Kinase Inhibitors; Quinolines; Signal Transduction; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Tissue Array Analysis; Xenograft Model Antitumor Assays

Aberrant protein glycosylation is involved in many diseases including cancer. This study investigated the role of fucosyltransferase VII (FUT7) in the progression of follicular thyroid carcinoma (FTC). FUT7 expression was found to be upregulated in FTC compared to paracancerous thyroid tissue, and in FTC with T2 stage of TMN classification compared to FTC with T1 stage. FUT7 overexpression promoted cell proliferation, epithelial-mesenchymal transition (EMT), and the migration and invasion of primary FTC cell line FTC-133. Consistently, FUT7 knock-down inhibited cell proliferation, EMT, as well as the migration and invasion of the metastatic FTC cell line FTC-238. Mechanistic investigation revealed that FUT7 catalyzed the α1,3-fucosylation of epidermal growth factor receptor (EGFR) in FTC cells. The extent of glycan α1,3-fucosylation on EGFR was positively correlated with the activation of EGFR in the presence/absence of epidermal growth factor (EGF) treatment. Furthermore, FUT7 was shown to enhance EGF-induced progression of FTC cells through mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathways. These findings provide a new perspective on FUT7 that may be a novel diagnostic and therapeutic target of FTC.

Topics: Adenocarcinoma, Follicular; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Fucosyltransferases; Humans; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Thyroid Neoplasms; Up-Regulation

Human follicular thyroid cancer cells (FTC-133) were sent to space via a sounding rocket during the TEXUS-53 mission to determine the impact of short-term microgravity on these cells. To enable cell culture and fixation in real microgravity, an automated experiment container (EC) was constructed. In order to ensure safe cell culture, cell-chambers consisting of polycarbonate (PC) material were used. They were highly biocompatible as proved by measuring cell survival using Annexin V flow cytometry. In the follow-up experiment, FTC-133 cells were sent to space via a sounding rocket and were fixed before and after the microgravity (µg) phase with RNAlater. In addition, cells were tested for reactions on hypergravity (hyper-g) as much as 18 g to determine whether worst case acceleration during launch can have an influence on the cells. We investigated genes belonging to biological processes such as cytoskeleton, cell adhesion, tumor growth, angiogenesis and apoptosis. Pathway analyses revealed central functions of VEGFA and EGF. EGF upregulates aspartate beta-hydroxylase (ASPH) which is influencing CASP3. Hyper-g induced a significant up-regulation of TUBB1, VIM, RDX, CAV1, VEGFA and BCL2. FTC-133 cells grown in an automated EC exposed to µg revealed moderate gene expression changes indicating their survival in orbit.

Topics: Biocompatible Materials; Calcium-Binding Proteins; Cell Line, Tumor; Cytoskeleton; Down-Regulation; Epidermal Growth Factor; Gene Expression; Humans; Hypergravity; Membrane Proteins; Mixed Function Oxygenases; Muscle Proteins; Proto-Oncogene Proteins c-bcl-2; Space Flight; Thyroid Neoplasms; Tubulin; Up-Regulation; Vascular Endothelial Growth Factor A; Weightlessness

Anaplastic thyroid cancer (ATC) is among the most aggressive types of malignant cancer. Epidermal growth factor (EGF) plays a crucial role in the pathogenesis of ATC, and patients with thyroid carcinoma typically exhibit increased cysteine-rich protein 61 (Cyr61). In this study, we found that EGF treatment induced cell migration, stress fiber formation, Cyr61 mRNA and protein expressions, and Cyr61 protein secretion in ATC cells. The recombinant Cyr61 protein significantly induced cell migration; however, inhibition of Cyr61 activity by a Cyr61-specific antibody abrogated EGF-induced cell migration. EGF treatment also affected epithelial-to-mesenchymal transition (EMT)-related marker protein expression, as evidenced by an increase in vimentin and a decrease in E-cadherin expression. Inhibition of Cyr61 expression by Cyr61 siRNA decreased cell migration and reversed the EMT-related marker protein expression. EGF treatment increased the phosphorylation of the extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB), and finally activated Cyr61 promoter plasmid activity. Our results suggest that Cyr61 is induced by EGF through the ERK/CREB signal pathway and that it plays a crucial role in the migration and invasion of ATC cells; moreover, Cyr61 might be a therapeutic target for metastatic ATC.

Topics: Cell Line, Tumor; Cell Movement; Cyclic AMP Response Element-Binding Protein; Cysteine-Rich Protein 61; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Humans; Phosphorylation; Signal Transduction; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms

This study was set to study the molecular mechanism underlying how miR-200 regulates EGF/EGFR signaling to involve in epithelial-mesenchymal transition (EMT) in anaplastic thyroid cancer (ATC) cells. Loss-of-function experiments of EGFR silencing by siRNA transfection was performed. Transfection of pre-miR-200s or anti-miR-200s was used to increase or decrease miR-200 transcripts. Real-time PCR, Western blot, immunohistochemistry, and transwell experiments were performed to determine the role of miR-200s in EMT and its role in EGF/EGFR-mediated EMT in vitro and in vivo. EGF/EGFR signaling activation increased the expression of mesenchymal marker vimentin in Nthy-ori 3-1 cells and decreased the expression of endothelial maker E-cadherin. EGF stimulation led to increased RhoA expression in Nthy-ori 3-1 cells. EGFR silencing resulted in decreased RhoA expression in SW1736 and ARO cells. EGF stimulation led to down-regulation of miR-200s and EMT. Restoration of miR-200 expression by pre-miR-200a/c transfection reversed the process, including increased E-cadherin and decreased vimentin. Down-regulation of miR-200 by anti-miR-200 effectively reduced miR-200. Matrigel invasion assay proved that restoration of miR-200 expression counteracted invasiveness. EGFR silencing decreased invasiveness in SW1736 cells, while down-regulation of miR-200s restored invasiveness. Xenograft tumors of SW1736 cells with cotransfection of anti-miR-200s and EGFR siRNA which kept the similar E-cadherin and vimentin expression with the untransfected controls. In ATC cells, miR-200s play a central role in EGF/EGFR-mediated invasiveness in vitro and EMT in vivo.

Topics: Antigens, CD; Cadherins; Cell Line, Tumor; Down-Regulation; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; MicroRNAs; Neoplasm Invasiveness; Neoplasm Transplantation; rhoA GTP-Binding Protein; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Vimentin

Our previous studies have demonstrated that epidermal growth factor (EGF) can induce cell migration through the induction of cysteine-rich protein 61 (Cyr61) in human anaplastic thyroid cancer (ATC) cells. The aim of the present study was to determine the inhibitory effects of combined treatment with the peroxisome proliferator-activated receptor-γ (PPARγ) ligand troglitazone and the cholesterol-lowering drug lovastatin at clinically achievable concentrations on ATC cell migration. Combined treatment with 5 μM troglitazone and 1 μM lovastatin exhibited no cytotoxicity but significantly inhibited EGF-induced migration, as determined using wound healing and Boyden chamber assays. Cotreatment with troglitazone and lovastatin altered the epithelial-to-mesenchymal-transition (EMT) -related marker gene expression of the cells; specifically, E-cadherin expression increased and vimentin expression decreased. In addition, cotreatment reduced the number of filopodia, which are believed to be involved in migration, and significantly inhibited EGF-induced Cyr61 mRNA and protein expression as well as Cyr61 secretion. Moreover, the phosphorylation levels of 2 crucial signal molecules for EGF-induced Cyr61 expression, the cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK), were decreased in cells cotreated with troglitazone and lovastatin. Performing a transient transfection assay revealed that the combined treatment significantly suppressed Cyr61 promoter activity. These results suggest that combined treatment with low doses of troglitazone and lovastatin effectively inhibits ATC cell migration and may serve as a novel therapeutic strategy for metastatic ATC.

Topics: Cell Line, Tumor; Cell Movement; Chromans; Cyclic AMP Response Element-Binding Protein; Cysteine-Rich Protein 61; Down-Regulation; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Humans; Lovastatin; Signal Transduction; Thiazolidinediones; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Troglitazone

Normal thyrocytes grown as reconstituted follicles in collagen gel were evaluated for drug effects of small molecule kinase inhibitors on growth factor-induced cell migration in a 3D context. MEK inhibition by U0126 only partially antagonized EGF/serum-induced cell migration from the basal follicular surface into the matrix. Combined treatment with U0126 and LY294002, a PI3K blocker, was necessary to abolish migration. However, exposure to only LY294002 facilitated the response to EGF by breakdown of the original follicular structure. In the same time EGF promoted thyroid cell survival that was compromised by LY294002 in absence of EGF. Cells treated with EGF and LY294002 retained the ability to form follicles. The findings indicate that dual inhibition of MAPK and PI3K/AKT pathways is required to fully block matrix invasion of EGF-stimulated thyroid cells. Conversely, single drug treatment with PI3K inhibitor adversely promotes invasiveness probably by destabilizing the follicular epithelium.

Topics: Animals; Butadienes; Cell Movement; Cell Survival; Chromones; Enzyme Inhibitors; Epidermal Growth Factor; Extracellular Matrix; MAP Kinase Signaling System; Morpholines; Neoplasm Invasiveness; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Swine; Thyroid Gland; Thyroid Neoplasms; Tissue Culture Techniques

The role of EGF and TGF-β1 in thyroid cancer is still not clearly defined. TGF-β1 inhibited the cellular growth and migration of follicular (FTC-133) and papillary (B-CPAP) thyroid carcinoma cell lines. Co-treatments of TGF-β1 and EGF inhibited proliferation in both cell lines, but displayed opposite effect on their migratory capability, leading to inhibition in B-CPAP and promotion in FTC-133 cells, by a MAPK-dependent mechanism. TGF-β1, TβRII and EGFR expressions were evaluated in benign and malignant thyroid tumors. Both positivity (51.7% and 60.0% and 80.0% in FA and PTC and FTC) and overexpression (60.0%, 77.7% and 75.0% in FA, PTC and FTC) of EGFR mRNA correlates with the aggressive tumor behavior. The moderate overexpression of TGF-β1 and TβRII mRNA in PTC tissues (61.5% and 62.5%, respectively), counteracted their high overexpression in FTC tissues (100% and 100%, respectively), while EGFR overexpression was similar in both carcinomas. Papillary carcinomas were positive to E-cadherin expression, while the follicular carcinomas lose E-cadherin staining. Our findings of TGF-β1/TβRII and EGFR overexpressions together with a loss of E-cadherin observed in human follicular thyroid carcinomas, and of increased migration ability MAPK-dependent after EGF/TGF-β1 treatments in the follicular thyroid carcinoma cell line, reinforced the hypothesis of a cross-talk between EGF and TGF-β1 systems in follicular thyroid carcinomas phenotype.

Topics: Adult; Aged; Cadherins; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression; Humans; Male; Middle Aged; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Thyroid Neoplasms; Transforming Growth Factor beta1; Young Adult

For thyroid tumorigenesis, two main human in vitro models are available: primary cultures of human thyrocytes treated with TSH or EGF/serum as models for autonomous adenomas (AA) or papillary thyroid carcinomas (PTC) respectively, and human thyroid tumor derived cell lines. Previous works of our group have assessed properties of those models, with a special emphasis on mRNA regulations. It is often assumed that miRNA may be one of the primary events inducing these mRNA regulations.. The purpose of this study was to investigate the representativity of those models to study microRNA regulations and their relation with mRNA expression. To achieve this aim, the miRNA expressions profiles of primary cultures treated with TSH or EGF/serum and of 6 thyroid cancer cell lines were compared to the expression profiles of 35 tumor tissues obtained by microarrays.. Our data on primary cultures have shown that the TSH or EGF/serum treatment did not greatly modify the microRNA expression profiles, which is contrary to what is observed for mRNA expression profiles, although they still evolved differently according to the treatment. The analysis of miRNA and mRNA expressions profiles in the cell lines has shown that they have evolved into a common, dedifferentiated phenotype, closer to ATC than to the tumors they are derived from.. Long-terms TSH or EGF/serum treatments do not mimic AA or PTC respectively in terms of miRNA expression as they do for mRNA, suggesting that the regulations of mRNA expression induced by these physiological agents occur independently of miRNA. The general patterns of miRNA expression in the cell lines suggest that they represent a useful model for undifferentiated thyroid cancer. Mirna probably do not mediate the rapid changes in gene expression in rapid cell biology regulation.

Topics: Carcinoma, Papillary; Cell Line, Tumor; Cell Transformation, Neoplastic; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Oligonucleotide Array Sequence Analysis; RNA, Messenger; Thyroid Gland; Thyroid Neoplasms; Thyrotropin

Poorly differentiated thyroid carcinomas are refractory to common anticancer therapies, and novel inhibitors are being tested in these deadly malignancies. The epidermal growth factor receptor (EGFR) tyrosine kinase represents an attractive target for treatment because it is up-regulated in thyroid cancer and plays a role in cancer progression. However, EGFR inhibitors have provided poor results in thyroid carcinomas.. We evaluated the possible mechanism underlying the resistance of thyroid cancer cells to EGFR inhibitors.. We tested the effect of the EGFR tyrosine kinase inhibitor gefitinib in a panel of thyroid cancer cell lines.. We found that in most of the cell lines, although gefitinib inhibited EGFR phosphorylation, it was poorly effective in reducing cell viability. gefitinib, however, was able to inhibit epidermal growth factor-induced cell migration and matrix invasion. In most thyroid cancer cell lines, gefitinib significantly inhibited Akt phosphorylation by inhibiting EGFR activation, but it had limited or no effect on ERK phosphorylation. The poor cell response to gefitinib was associated with genetic alterations, leading to constitutive activation of the ERK pathway, including BRAF(V600E) and HRAS(G12A/Q61R) mutations and RET/PTC1 rearrangement. When BRAF(V600E)-positive thyroid cancer cells were incubated with the specific BRAF inhibitor PLX4032, sensitivity to gefitinib was restored. Similar results were obtained with rat sarcoma and RET/papillary thyroid cancer inhibitors.. These results indicate that thyroid cancer resistance to gefitinib is due to the constitutive activation of the mitogenic pathway by either signals downstream of EGFR or other tyrosine kinase receptors. This resistance can be overcome by the combined use of selective inhibitors.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Genes, ras; Humans; MAP Kinase Signaling System; Mutation; Neoplasm Invasiveness; Phosphorylation; Proto-Oncogene Proteins B-raf; Quinazolines; Thyroid Neoplasms

In patients without metastases, capsular and vascular invasion must be noted to make the diagnosis of follicular thyroid carcinoma (FTC). Some patients are initially diagnosed as follicular adenoma (FA) but develop metastases, indicating the original lesion was FTC. A diagnostic marker for FTCs that appear to be FAs by conventional histopathology is urgently needed. CD147 is a transmembrane glycoprotein that induces matrix metalloproteinases (MMPs) and participates in carcinoma invasion. The objective of this study was to determine whether CD147 is upregulated in FTC and if measures directed against it could reduce the invasive activity of FTC cells.. The expression levels of CD147, MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9 in surgical specimens of normal thyroid (n=8), FA (n=20), and FTC (n=9) was determined using immunoblot and immunohistochemical techniques. CD147 protein expression levels of epithelial growth factor stimulated FTC-133 cell lines was measured by immunoblotting with and without cell signaling inhibitors such as wortmannin, PD98059, SP600125, and SB203580. This was also done after exposure to short-hairpin interference RNA directed against CD147.. Immunoblot analysis of thyroid tissues revealed significant increases in signals for CD147, MMP-3, MMP-7, and MMP-9 in FTC compared with FA or normal tissue, or both. Immunohistochemical analysis revealed colocalization of determinants of CD147 with those of all of MMPs studied, mainly in follicular cells in normal and neoplastic cells in FA and FTC; their immunoreactivities were to some extent more intense in the FTC than FA or normals. In FTC-133 cells, immunoreactive signals for CD147 were upregulated by epidermal growth factor (EGF), and the EGF-driven increases in CD147 were prevented by inhibitors against phosphoinositol-3 kinase (PI3K), extracellular signal-regulated protein kinase (ERK), or c-Jun N-terminal kinase (JNK) but not p38. RNA interference targeted against CD147 reduced the invasive activity of FTC-133 cells and was associated with downregulation of MMP-2, MMP-3, MMP-7, and MMP-9.. These results provide in vivo evidence for CD147 upregulation in FTC and in vitro evidence for EGF-stimulated CD147 induction via the PI3K, ERK, and JNK pathways. They suggest the involvement of CD147 in the invasiveness of FTC cells via regulation of MMPs.

Topics: Adenocarcinoma, Follicular; Adenoma; Adolescent; Adult; Aged; Basigin; Blotting, Western; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Female; Gene Silencing; Humans; Immunohistochemistry; Male; Matrix Metalloproteinases; Middle Aged; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; Thyroid Neoplasms; Young Adult

The miR-200 family was recently identified as a suppressor of epithelial-mesenchymal transition (EMT). The loss or gain of miR-200 family members is associated with cancer invasion. The epidermal growth factor receptor (EGFR) is overexpressed in the majority of anaplastic thyroid cancers (ATCs). The activation of EGFR by its ligand, epidermal growth factor (EGF), activates a signaling cascade that results in the enhanced migration and invasiveness of thyroid cancer cells. However, little is known about the potential interrelationships between EGF/EGFR, miR-200s and the induction of EMT or mesenchymal-epithelial transition (MET) processes. This study aimed to investigate the regulatory role of miR-200s in EMT modulation by EGF/EGFR. Using transfection, real-time reverse transcription PCR and western blot analysis, we found that the EGF treatment of Nthy-ori 3-1 thyroid follicular cells resulted in the downregulation of E-cadherin and the upregulation of vimentin. By contrast, EGFR silencing in SW1736 human thyroid carcinoma cells led to the upregulation of E-cadherin and the downregulation of vimentin. In addition, EGF signaling correlated with the reduced expression of miR-200s and the re-expression of miR-200s abrogated the effects of EGF treatment and restored an epithelial phenotype to EGF-induced Nthy-ori 3-1 cells. Conversely, the silencing of miR-200s in SW1736 cells overcame siEGFR-induced changes in gene expression and phenotype. In addition, we demonstrate that miR-200s play a key role in in vitro EGF/EGFR-mediated thyroid cell invasion and in EMT in vivo. We, therefore, provide a mechanistic link between the miR-200 family and EGF/EGFR, which suggests that miR-200 upregulation may serve as a novel therapeutic strategy for highly invasive thyroid cancers.

Topics: Animals; Cadherins; Cell Line; Cell Line, Tumor; Down-Regulation; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Mice; MicroRNAs; Neoplasm Invasiveness; rhoA GTP-Binding Protein; RNA Interference; Thyroid Carcinoma, Anaplastic; Thyroid Gland; Thyroid Neoplasms; Vimentin

Anaplastic thyroid carcinoma (ATC) is one of the most lethal human cancers with a median survival of 6 months. The inhibition of epidermal growth factor receptor (EGFR) alone, or with VEGF receptor 2 (VEGFR2), represents an attractive approach for treatment of ATC. Several reports have examined agents that target these receptors. However, with the misidentification of as many as 60% of all commonly used ATC cell lines, the significance of these past findings is unclear.. Cell lines authenticated by short tandem repeat profiling were selected to establish xenograft tumors in an orthotopic murine model of ATC. These mice were then treated with vandetanib to evaluate its effects on ATC tumor growth. Dynamic contrast-enhanced (DCE) MRI was utilized to measure the impact of vandetanib on tumor vasculature.. Vandetanib inhibited tumor growth of the ATC cell lines Hth83 and 8505C in vivo by 69.3% (P < 0.001) and 66.6% (P < 0.05), respectively, when compared with control. Significant decreases in vascular permeability (P < 0.01) and vascular volume fraction (P < 0.05) were detected by DCE-MRI in the orthotopic xenograft tumors after 1 week of treatment with vandetanib as compared with control.. The inhibition of EGFR and VEGFR2 by vandetanib and its tremendous in vivo antitumor activity against ATC make it an attractive candidate for further preclinical and clinical development for the treatment of this particularly virulent cancer, which remains effectively untreatable. Vandetanib disrupts angiogenesis and DCE-MRI is an effective method to quantify changes in vascular function in vivo.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Humans; Immunoblotting; In Situ Nick-End Labeling; Magnetic Resonance Imaging; Male; Mice; Mice, Nude; Neovascularization, Pathologic; Phosphorylation; Piperidines; Platelet Endothelial Cell Adhesion Molecule-1; Quinazolines; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Tumor Burden; Vascular Endothelial Growth Factor Receptor-2; Xenograft Model Antitumor Assays

Topics: Adenocarcinoma, Papillary; Aged; Antineoplastic Agents; Carcinoma, Signet Ring Cell; DNA Mutational Analysis; Epidermal Growth Factor; Erlotinib Hydrochloride; Exons; Female; Humans; Immunohistochemistry; Mutation; Polymerase Chain Reaction; Quinazolines; Thyroid Neoplasms

Mitogen-inducible gene 6 (Mig-6) is a putative tumor suppressor gene and prognostic biomarker in papillary thyroid cancer. We hypothesized that Mig-6 knockout would activate pro-oncogenic signaling in mouse thyrocytes.. We performed a thyroid-specific knockout using the Cre/loxP recombinase system.. Four knockout and 4 control mouse thyroids were harvested at 2 months of age. Immunoblotting confirmed Mig-6 ablation in knockout mice thyrocytes. Epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK) phosphorylation levels were increased in Mig-6 knockout compared to wild-type mice. Total EGFR levels were similar in knockout and wild-type mice. However, EGFR was absent in the caveolae-containing membrane fraction of knockout mice, indicating that Mig-6 depletion is associated with a change in the membrane distribution of EGFR. Although p65 localized to the nucleus in wild-type mice, it was distributed in both cytoplasm and nucleus in knockouts, suggesting that Mig-6 loss decreases p65 activity.. Our results confirm the feasibility of targeted, thyroid-specific gene knockout as a strategy for studying the relevance of specific genes in thyroid oncogenesis. We suggest that the loss of Mig-6 alters the membrane distribution of EGFR, which may limit receptor degradation and activate this oncogenic signaling pathway.

Topics: Adaptor Proteins, Signal Transducing; Animals; Biomarkers; Carcinoma; Carcinoma, Papillary; Epidermal Growth Factor; Gene Knockout Techniques; Genes, Tumor Suppressor; Intracellular Signaling Peptides and Proteins; Mice; Mice, Knockout; NF-kappa B; Signal Transduction; Thyroid Cancer, Papillary; Thyroid Gland; Thyroid Neoplasms; Transcription Factor RelA

Proteomics may help to elucidate differential signaling networks underlying the effects of compounds and to identify new therapeutic targets. Using a proteomic-multiplexed analysis of the phosphotyrosine signaling together with antibody-based validation techniques, we identified several candidate molecules for RET (rearranged during transfection) tyrosine kinase receptor carrying mutations responsible for the multiple endocrine neoplasia type 2A and 2B (MEN2A and MEN2B) syndromes in two human medullary thyroid carcinoma (MTC) cell lines, TT and MZ-CRC-1, which express the RET-MEN2A and RET-MEN2B oncoproteins, respectively. Signaling elements downstream of these oncoproteins were identified after treating cells with the indolinone tyrosine kinase inhibitor RPI-1 to knock down RET phosphorylation activity. We detected 23 and 18 affinity-purified phosphotyrosine proteins in untreated TT and MZ-CRC-1 cells, respectively, most of which were shared and sensitive to RPI-1 treatment. However, our data clearly point to specific signaling features of the RET-MEN2A and RET-MEN2B oncogenic pathways. Moreover, the detection of high-level expression of minimally phosphorylated epidermal growth factor receptor (EGFR) in both TT and MZ-CRC-1 cells, together with our data on the effects of EGF stimulation on the proteomic profiles and the response to Gefitinib treatment, suggest the relevance of EGFR signaling in these cell lines, especially since analysis of 14 archival MTC specimens revealed EGFR mRNA expression in all samples. Together, our data suggest that RET/EGFR multi-target inhibitors might be beneficial for therapy of MTC.

Topics: Animals; Antineoplastic Agents; Carcinoma, Medullary; Epidermal Growth Factor; ErbB Receptors; Female; Gefitinib; Germ-Line Mutation; Humans; Mice; Mice, Nude; Multiple Endocrine Neoplasia Type 2a; Multiple Endocrine Neoplasia Type 2b; Oncogene Proteins; Phosphorylation; Proteomics; Proto-Oncogene Proteins c-ret; Quinazolines; Signal Transduction; Thyroid Neoplasms; Tyrosine

The intracellular domains of the membrane-anchoring regions of some precursors of epidermal growth factor (EGF) family members have intrinsic biologic activities. We have determined the role of the human proEGF cytoplasmic domain (proEGFcyt) as part of the proEGF transmembrane-anchored region (proEGFctF) in the regulation of motility and elastinolytic invasion in human thyroid cancer cells. We found proEGFctF to act as a negative regulator of motility and elastin matrix penetration and the presence of proEGFcyt or proEGF22.23 resulted in a similar reduction in motility and elastinolytic migration. This activity was counteracted by EGF-induced activation of EGF receptor signaling. Decreased elastinolytic migratory activity in the presence of proEGFctF and proEGFcyt/proEGF22.23 coincided with decreased secretion of elastinolytic procathepsin L. The presence of proEGFctF and proEGFcyt/proEGF22.23 coincided with the specific transcriptional up-regulation of t-SNARE member SNAP25. Treatment with siRNA-SNAP25 resulted in motility and elastin migration being restored to normal levels. Epidermal growth factor treatment down-regulated SNAP25 protein by activating EGF receptor-mediated proteasomal degradation of SNAP25. These data provide first evidence for an important function of the cytoplasmic domain of the human proEGF transmembrane region as a novel suppressor of motility and cathepsin L-mediated elastinolytic invasion in human thyroid carcinoma cells and suggest important clinical implications for EGF-expressing tumors.

Topics: Animals; Carcinoma; Cathepsin L; Cathepsins; Cell Movement; Cysteine Endopeptidases; Cytoplasm; Down-Regulation; Elastin; Epidermal Growth Factor; Female; Humans; Hydrolysis; Male; Models, Biological; Neoplasm Invasiveness; Protein Precursors; Protein Structure, Tertiary; Thyroid Neoplasms; Transfection; Tumor Cells, Cultured

In thyroid cancer (TC) endostatin was identified as a powerful negative regulator of tumor angiogenesis in vitro. It is currently being evaluated in phase I trials for antiangiogenic therapy in various solid tumors. The aim of this study was to evaluate endostatin expression in archival TC specimens and its secretion following stimulation with thyrotropin (TSH) and epidermal growth factor (EGF) in TC cell lines.. Tissue microarrays of 44 differentiated and 7 anaplastic TC and their metastasis were immunostained for endostatin protein expression and compared with corresponding non-neoplastic thyroid tissue (NT). In vitro, six differentiated (FTC133, FTC236, HTC, HTC-TSHr, XTC, and TPC1) and three anaplastic (C643, Hth74, Kat4.0) TC cell lines were evaluated for basal as well as TSH (1-100 mU/ml) and EGF stimulated (1-100 ng/ml) endostatin.. Endostatin was detected in all TC and more than half of the NT. Endostatin expression was more frequent and intense in differentiated as compared to anaplastic TC. In vitro, basal endostatin secretion varied between 33 +/- 5 pg/ml (FTC236) and 549 +/- 65 pg/ml (TPC1) and was doubled in FTC, when the "primary" (FTC133) was compared with the metastasis (FTC236). Some cell lines showed TSH-induced (e.g., 60% in XTC) or EGF-induced (e.g., 120% in TPC1) upregulation of endostatin secretion, while others did not, despite documented receptor expression.. This study demonstrates endostatin expression in TC, metastasis and--less frequently and intensely--in NT, suggesting a possible association to tumor progression. In vitro, endostatin secretion of some cell lines is regulated by TSH and EGF, however the individual differences deserve further functional studies. These results support rather tumor-specific than histotype-specific expression and regulation of endostatin in TC.

Topics: Adenocarcinoma, Follicular; Angiogenesis Inhibitors; Carcinoma; Carcinoma, Papillary; Cell Differentiation; Endostatins; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Humans; Immunoenzyme Techniques; Lymphatic Metastasis; Paraffin Embedding; Thyroid Gland; Thyroid Neoplasms; Thyrotropin; Tumor Cells, Cultured

In order to further advance our knowledge of the role epidermal growth factor (EGF) plays in thyroid carcinoma, we investigated its effect on the regulation of matrix metalloproteinase-9 (MMP-9), a key enzyme that plays an important role in tumor invasion and angiogenesis. The expression of MMP-9 in EGF-treated and untreated human follicular thyroid carcinoma cells (FTC-133) was evaluated using reverse transcription-PCR, Western blot and gelatin zymography. Transient transfection and electrophoretic mobility shift assays (EMSA) were also performed to measure MMP-9 promoter activity, to identify multiple signaling pathways and to determine a proximal AP-1-binding site located between -79 to -73 base pairs upstream of the transcriptional start site that is involved in activation of MMP-9 by EGF. In the present study, we demonstrate that EGF treatment up-regulated MMP-9 expression in human follicular thyroid carcinoma cells. Expression of FAK-related non kinase (FRNK), a potent dominant-negative inhibitor of FAK, reduced FAK auto-phosphorylation and inhibited EGF-induced MMP-9 transcription and secretion leading to decreased cell invasion through Matrigel in in vitro Transwell assays. Our studies highlight the role FAK plays in promoting cell invasion through the activation of distinct signaling pathways induced by EGF with protein MMP-9 transcription and secretion in follicular thyroid carcinoma cells.

Topics: Adenoma; Binding Sites; Cell Line, Tumor; Epidermal Growth Factor; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Proteins; Promoter Regions, Genetic; Signal Transduction; Thyroid Neoplasms; Transcription Factor AP-1

Constitutive activation of the RAS/RAF/MAPK pathway has been found in different tumor types including papillary thyroid carcinomas (PTCs). To get more insight into genes primarily regulated in the human tumor cells, an in vitro model was developed in which primary cultures of human thyrocytes were treated for different times with epidermal growth factor and serum (EGF/serum), which stimulate the MAPK cascade. Gene expression profiles were obtained by microarrays and compared to the expression profiles of PTCs. An evolution from short-term to long-term EGF/serum-treated cells was found, i.e., a program change showing a distinction between gene expression profiles of short-term and long-term EGF/serum-treated cells. The late pattern of EGF/serum stimulated cells converges to the pattern of PTCs. Comparison of these two types of cells with cAMP activated cells, from thyroid-stimulating hormone-treated thyrocytes and autonomous adenomas, showed distinct gene expression profiles for the two pathways. For the two models, an overlap was found in a number of genes which were early induced in vitro but down-regulated later in vitro and in the in vivo tumors. Thus, long-term stimulated human primary cultures demonstrate a clear relation with the tumor in vivo and could therefore be used as models for the disease.

Topics: Adult; Carcinoma, Papillary; Cell Transformation, Neoplastic; Cells, Cultured; Cyclic AMP; Epidermal Growth Factor; Humans; Middle Aged; Oligonucleotide Array Sequence Analysis; Serum; Thyroid Gland; Thyroid Neoplasms

This study evaluated the role of EGF and the effects of EGF-targeting drugs (Cetuximab, AEE 788) on growth, apoptosis, and autocrine VEGF-secretion of thyroid cancer (TC) cells. Autocrine activation of the epidermal growth factor receptor (EGF-R) is commonly regarded to contribute to the malignant phenotype of TC cells and may therefore represent a rational therapeutic target. Out of a number of TC cell lines two anaplastic (Hth74, C643), one follicular (FTC133), and one papillary thyroid cancer cell line (TPC1) were analyzed in depth for VEGF-R-and EGF-R-expression, basal and EGF-stimulated (1-100 ng/ml) VEGF protein secretion and proliferation. Subsequently the antiprolifereative and antiangiogenic effect of cetuximab (Erbitux), a monoclonal antibody that blocks the EGF-R and AEE 788, a novel dual-kinase inhibitor of EGF-R and VEGF-R were assessed, and the downstream EGF-R signal transduction was analyzed by means of detecting phosphorylated pEGF-R, pVEGF-R, pAkt, and p-MAPK. EGF stimulated VEGF-mRNA expression and protein secretion in all TC cell lines. The EGF-R antagonist Cetuximab consistently decreased VEGF secretion in all TC cell lines (min. 15%, n.s. in C643 cells and max. 90% in Hth74 cells, P < 0.05), but did not affect tumor cell proliferation in vitro. In contrast, the EGF-R- and VEGF-R-kinase inhibitor AEE 788 not only reduced VEGF secretion (min. 55%, P < 0.05 in C643 and max. 75%, P < 0.05, in FTC133), but also exhibited a dose-dependent inhibition of tumor cell proliferation (min. 75%, P < 0.05 in C643 and max. 95%, P < 0.05 in Hth74) and was a potent inductor of apoptosis in two of four TC cell lines. These effects were always accompanied by reduced levels of pEGF-R, pVEGF-R, pAkt, and pMAPK. Although inhibition of the EGF-receptor by Cetuximab potently disrupts autocrine secretion of VEGF, only the concurrent inhibition of the VEGF- and EGF receptor, e.g., by AEE 788 induces reduced proliferation and apoptosis in vitro. This suggests a particular rationale for the use of tyrosine kinase inhibitors with dual modes of action such as AEE 788 in thyroid cancer.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cetuximab; Drug Evaluation, Preclinical; Epidermal Growth Factor; ErbB Receptors; Humans; Purines; Receptors, Vascular Endothelial Growth Factor; Signal Transduction; Thyroid Neoplasms; Vascular Endothelial Growth Factor A

Mechanisms of invasion in thyroid cancer remain poorly understood. We hypothesized that signaling via the epidermal growth factor receptor (EGFR) stimulates thyroid cancer cell invasion by altering the expression and cleavage of matrix metalloproteinases (MMPs). Papillary and follicular carcinoma cell lines were treated with EGF, the EGFR tyrosine kinase inhibitor AG1478, and the MMP inhibitors GM-6001 and Col-3. Flow cytometry was used to detect EGFR. In vitro invasion assays, gelatin zymography, and quantitative reverse transcription-PCR were used to assess the changes in invasive behavior and MMP expression and activation. All cell lines were found to overexpress functional EGFR. EGF stimulated invasion by thyroid cancer cells up to sevenfold (P<0.0001), a process that was antagonized completely by AG1478 and Col-3, partially by GM-6001, but not by the serine protease inhibitor aprotinin. EGF upregulated expression of MMP-9 (2.64- to 8.89-fold, P<0.0001) and membrane type-1 MMP (MT1-MMP, 1.97- to 2.67-fold, P<0.0001). This effect was blocked completely by AG1478 and partially by Col-3. The activation of MMP-2 paralleled MT1-MMP expression. We demonstrate that MMPs are critical effectors of invasion in the papillary and follicular thyroid cancer cell lines studied. Invasion is regulated by signaling through EGFR, an effect mediated by augmentation of gelatinase expression and activation. MMP inhibitors and growth factor antagonists may be effective tumoristatic agents for the treatment of aggressive thyroid carcinomas.

Topics: Adenocarcinoma, Follicular; Aprotinin; Blotting, Western; Carcinoma, Papillary; Cell Differentiation; Dipeptides; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Neoplasm Invasiveness; Protease Inhibitors; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatases; Quinazolines; Reverse Transcriptase Polymerase Chain Reaction; Serine Proteinase Inhibitors; Tetracyclines; Thyroid Neoplasms; Tumor Cells, Cultured; Tyrphostins

Vascular endothelial growth factor (VEGF) induces proliferation of endothelial cells, stimulates angiogenesis and increases vascular permeability. Epidermal growth factor (EGF) induces proliferation of epidermal cells and stimulates epidermal migration. Increased VEGF and EGF expression have been associated with poor clinical outcome in many malignancies. Several recent reports have shown overexpression of VEGF and EGF in papillary thyroid cancer (PTC). The study aimed to determine the intensity of expression of VEGF and EGF in patients with PTC and to find any correlation between the intensity of the expression and staging of the disease at the moment of surgery. The study comprised a group of 48 consecutive patients with PTC who underwent radical surgery. The group consisted of 11, 25, eight and four patients at pT1N0M0, pT2N0M0, pT3N1M0 and pT4N1M0 stages, respectively. The control group was composed of 20 healthy bone marrow transplant donors, age-, gender- and BMI-matched with PTC patients. The immuno-chemiluminescence enzyme linked immunoassay (ELISA) method was used to determine the expression and level of VEGF and EGF in serum samples. Patients' characteristics, serum levels of VEGF and EGF, final histology and pTNM were analysed.. The mean serum level of VEGF and EGF was remarkably higher in PTC patients than in controls. A negative correlation between staging in pTNM classification and the mean serum VEGF level (r=-0.5168; P<0.05) as well as a positive correlation between staging in pTNM classification and the mean serum EGF level (r=0.6104; P<0.05) in PTC patients was observed.. Both VEGF and EGF play an important role in PTC growth. However, the intensity of their expression is variable, depending on the stage of the disease. The highest intensity of VEGF expression is characteristic for low-stage T1N0M0 disease, whereas the highest intensity of EGF expression is more often found at locally advanced pT3 and pT4N1M0 stages of the disease. Further long-term follow-up studies are required to demonstrate the prognostic value of VEGF and EGF in PTC, particularly in identification of patients with expected poor prognosis or shorter recurrence-free survival.

Topics: Adenocarcinoma, Papillary; Adult; Aged; Aged, 80 and over; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Male; Middle Aged; Neovascularization, Physiologic; Prospective Studies; Thyroid Neoplasms; Vascular Endothelial Growth Factor A

Thrombospondin-1 (TSP-1) is a multidomain extracellular macromolecule that was first identified as natural modulator of angiogenesis and tumor growth. In the present study, we found that epidermal growth factor (EGF) up-regulated TSP-1 expression in FTC-133 (primary tumor) but not in FTC-238 (lung metastasis) thyroid cancer cells. Both EGF and TSP-1 induced expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In FTC-133 cells, EGF induced proliferation in a TSP-1- and TIMP-1-dependent manner. In addition, we determined that re-expression of the tumor suppressor protein PTEN induced cell death, an effect that correlated with a block of Akt kinase phosphorylation. EGF-induced TSP-1 and TIMP-1 promoter activity and protein expression were inhibited in FTC-133 cells stably expressing wtPTEN but not in cells expressing mutant PTEN. Furthermore, we found that wtPTEN inhibited EGF--but not TSP-1--stimulated FTC-133 cell migration and also inhibited invasion induced by EGF and by TSP-1. Finally, an antibody against TSP-1 reversed EGF-stimulated FTC-133 cell invasion as well as the constitutive invasive potential of FTC-238 cells. Overall, our results suggest that PTEN can function as an important modulator of extracellular matrix proteins in thyroid cancer. Therefore, analyzing differential regulation of TSP-1 by growth factors such as EGF can be helpful in understanding thyroid cancer development.

Topics: Apoptosis; Carcinoma; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Humans; MAP Kinase Kinase 1; Phosphatidylinositol 3-Kinases; Phosphoric Monoester Hydrolases; Promoter Regions, Genetic; PTEN Phosphohydrolase; Thrombospondin 1; Thyroid Neoplasms; Tissue Inhibitor of Metalloproteinase-1; Tumor Suppressor Proteins; Up-Regulation

We studied the distribution of transcripts encoding the cytoplasmic domain of the membrane-anchored precursor epidermal growth factor (proEGFcyt) and a novel cytoplasmic proEGF splice isoform with a deleted exon 23 and an out-of-frame fusion of exon 24 (proEGFdel23) in human normal and neoplastic thyroid tissues. In papillary thyroid carcinoma (PTC), coexpression of transcripts encoding for both proEGFcyt and proEGFdel23 correlated with poor differentiation of PTC. To determine potential roles of the cytoplasmic proEGF domain in human thyroid cells, we generated stable transfectants of the human follicular thyroid carcinoma cell line FTC-133 overexpressing the normal cytoplasmic domain proEGFcyt, a truncated proEGFcyt composed of the peptide sequence encoded by exons 22 and 23 (proEGF22.23) and proEGFdel23. The proEGFcyt and proEGF22.23 transfectants displayed significantly reduced proliferation rates, an enlarged cellular phenotype, and alterations in the distribution and post-translational modification of the microtubular system. These transfectants also displayed increased production of microtubule-associated proteins 1b and 2c, which was absent in FTC-133-proEGFdel23 or FTC-133-empty plasmid transfectants. This is the first evidence of an involvement of proEGF cytoplasmic domain in microtubular stability in the human thyroid carcinoma cell line FTC-133 and may suggest a specific role for the cytoplasmic domain of membrane-anchored proEGF, particularly exon 23, in thyroid carcinoma. The up-regulation of proEGFdel23 in poorly differentiated PTC and the exclusive detection of both proEGF isoforms in undifferentiated thyroid carcinoma may indicate an involvement of this novel truncated proEGFdel23 cytoplasmic domain during dedifferentiation processes of human thyroid cells.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Base Sequence; Carcinoma, Papillary; Child; Cytoplasm; Epidermal Growth Factor; Female; Humans; Male; Microtubule-Associated Proteins; Microtubules; Middle Aged; Molecular Sequence Data; Neoplasm Staging; Protein Isoforms; Protein Precursors; Protein Processing, Post-Translational; Protein Structure, Tertiary; Thyroid Neoplasms; Transfection; Tubulin

We investigated integrin-linked kinase (ILK), a focal adhesion serine-threonine protein kinase, as a new molecular target for treating anaplastic thyroid cancer. ILK mediates cell growth and survival signals and is overexpressed in a number of cancers. Therefore, we hypothesized that inhibition of ILK leads to growth arrest and apoptosis of thyroid cancer cells. According to Western blotting, the level of ILK protein was highly expressed in one papillary (NPA187) and four of five (Hth74, DRO, ARO, KAT4, and K4) anaplastic thyroid cancer cell lines. Immunohistochemical analysis of a human tissue microarray revealed that ILK was highly expressed in anaplastic thyroid cancer but not in normal human thyroid tissue. Treating thyroid cancer cell lines with a new ILK inhibitor, QLT0267, inhibited epidermal growth factor-induced phosphorylation of AKT, inhibited cell growth, and induced apoptosis in the NPA187, DRO, and K4 cell lines. QLT0267 also inhibited the kinase activity of immunoprecipitated ILK in four of five cell lines. Tumor volumes in mice treated with QLT0267 were significantly reduced compared with those in untreated mice. In immunohistochemical studies, QLT0267 suppressed phosphorylated p-AKT and angiogenesis (i.e., reduced mean vascular density) and induced apoptosis in both tumor cells and tumor-associated endothelial cells of the thyroid DRO xenografts. In summary, we found that ILK expression and activity were elevated in human anaplastic thyroid cancer and ILK inhibition led to growth arrest and apoptosis in vitro and in vivo. Our results provide preliminary evidence that ILK is a potential therapeutic target for treating anaplastic thyroid cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Endothelial Cells; Epidermal Growth Factor; Humans; Male; Mice; Neovascularization, Pathologic; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Thyroid Neoplasms; Tissue Array Analysis; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Autotaxin (ATX/NPP2) is a tumor cell motility-stimulating factor that displays both a nucleotide pyrophosphatase/phosphodiesterase activity and a recently described lysophospholipase D (lysoPLD) activity. The precise function of ATX in tumor cells and the role of ATX in thyroid carcinoma remains unclear. We have quantified ATX mRNA expression in thyroid carcinoma cell lines and in tissues of patients with thyroid carcinomas. ATX gene activity was significantly higher in undifferentiated anaplastic thyroid carcinoma cell lines (UTC) and tumor tissues as compared to follicular thyroid carcinoma (FTC) cell lines, FTC tissues or goiter tissues that were used as a control. In the thyroid carcinoma cell line 1736, EGF and bFGF stimulated ATX mRNA expression, whereas the cytokines IL-4, IL-1beta and TGF-beta reduced ATX transcriptional levels. FTC-133 cells, stably transfected with an expression vector for ATX, showed a higher lysoPLD activity, a higher proliferation rate and an increased migratory behavior. In addition, ATX also displayed a paracrine stimulatory effect on the motility of different thyroid carcinoma cell lines. Overexpression of ATX in the stably transfected FTC-133 resulted in down-regulation of CD54/ intercellular adhesion molecule-1 (ICAM-1) gene expression and augmented gene activity of the pro-angiogenic chemokine IL-8. We conclude that ATX may be regarded as a new tissue marker for undifferentiated human thyroid carcinoma cells. ATX increases the proliferation and migration of thyroid carcinoma cell lines and may also affect the angiogenic potential of thyroid carcinoma cells. Further studies are needed to provide insight into the role of ATX in the normal and neoplastic thyroid gland.

Topics: Adenocarcinoma, Follicular; Adult; Aged; Aged, 80 and over; Carcinoma; Carcinoma, Papillary; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Glucose-6-Phosphate Isomerase; Glycoproteins; Goiter; Humans; Intercellular Adhesion Molecule-1; Interleukin-1; Interleukin-4; Interleukin-8; Male; Middle Aged; Multienzyme Complexes; Phosphodiesterase I; Phosphoric Diester Hydrolases; Pyrophosphatases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thyroid Neoplasms; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

The activity of thymidine kinase (TK-EC 2.7.1.21)--an enzyme functioning as a part of the pyrimidine salvage pathway of DNA synthesis--is closely related to growth processes. The aim of the study was to measure TK activity in homogenates of human thyroid tissue of the following types: non-toxic nodular goiter (NTNG)--macroscopically unchanged tissue, non-toxic adenoma (NTA), and toxic adenoma (TA) (obtained from patients, who--before the surgery--had been treated with thyrostatic drugs for thyrotoxicosis). Thyroid tissue was obtained from female patients subjected to subtotal thyroidectomy at the Department of Endocrine Surgery, Medical University of Lódź. Thyroid homogenates were incubated in the presence of epidermal growth factor (EGF), used in five different concentrations (0.1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml). TK activity was estimated by chromatographic measurements of the amount of the main reaction product--deoxythymidine monophosphate.. 1) We did not observe any significant difference between TK activity in the homogenates of the thyroid tissue collected from NTNG and NTA; TK activity was clearly higher in the homogenates of adenomatous tissue, collected from the patients with TA; 2) EGF increased TK activity in the homogenates of the macroscopically unchanged tissue, collected--during surgery--from the patients with NTNG, as well as in homogenates of thyroid tissue from NTA; 3) In case of hyperactive thyroid tissue, obtained from TA, EGF tended to increase TK activity, however, without any statistical differences. Our results confirm TK increased activity in hyperactive thyroid tissue. At the same time, the obtained data suggest a certain role of EGF in goiter formation in humans.

Topics: Adenoma, Oxyphilic; Animals; Enzyme Activation; Epidermal Growth Factor; Female; Goiter, Nodular; Humans; Thymidine Kinase; Thyroid Neoplasms

No effective treatment options currently are available to patients with anaplastic thyroid cancer (ATC), resulting in high mortality rates. Epidermal growth factor (EGF) has been shown to play a role in the pathogenesis of many types of cancer, and its receptor (EGFR) provides an attractive target for molecular therapy.. The expression of EGFR was determined in ATC in vitro and in vivo and in human tissue arrays of ATC. We assessed the potential of the EGFR inhibitor gefitinib ("Iressa," ZD1839) to inhibit EGFR activation in vitro and in vivo, inhibit ATC cellular proliferation, induce apoptosis, and reduce the growth of ATC cells in vivo when administered alone and in combination with paclitaxel.. EGFR was overexpressed in ATC cell lines in vitro and in vivo and in human ATC specimens. Activation of EGFR by EGF was blocked by the addition of gefitinib. In vitro studies showed that gefitinib greatly inhibited cellular proliferation and induced apoptosis in ATC cell lines and slowed tumor growth in a nude mouse model of thyroid carcinoma cells injected subcutaneously.. ATC cells consistently overexpress EGFR, rendering this receptor a potential target for molecular therapy. Gefitinib effectively blocks activation of EGFR by EGF, inhibits ATC cellular proliferation, and induces apoptosis in vitro. Our in vivo results show that gefitinib has significant antitumor activity against ATC in a subcutaneous nude mouse tumor model and therefore is a potential candidate for human clinical trials.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma; Carcinoma, Papillary; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Mice; Mice, Nude; Paclitaxel; Phosphorylation; Quinazolines; Thyroid Neoplasms; Transforming Growth Factor alpha; Tumor Cells, Cultured

Heat shock protein 90 (HSP90) serves as a chaperone protein and plays a critical role in tumor cell growth and/or survival. Geldanamycin, a specific inhibitor of HSP90, is cytotoxic to several human cancer cell lines, but its effect in thyroid cancer is unknown. We, therefore, investigated the effect of geldanamycin on cell proliferation, oncoprotein expression, and invasion in human thyroid cancer cell lines. We used six thyroid cancer cell lines: TPC-1 (papillary), FTC-133, FTC-236, FTC-238 (follicular), XTC-1 (Hürthle cell), and ARO (anaplastic). We used the dimethyl-thiazol-diphenyltetrazolium bromide assay, a clonogenic assay, an apoptotic assay, and a Matrigel invasion assay. We evaluated oncoprotein expression using Western blots and flow cytometry. After 6 d of treatment with 50 nM geldanamycin, the percent inhibition of growth was 29.4% in TPC-1, 97.5% in FTC-133, 96.7% in FTC-236, 10.8% in FTC-238, 70.9% in XTC-1, and 45.5% in ARO cell lines. In the FTC-133 cell line, geldanamycin treatment decreased clonogenicity by 21% at a concentration of 50 nM; geldanamycin induced apoptosis and down-regulated c-Raf-1, mutant p53, and epidermal growth factor (EGF) receptor expression; geldanamycin inhibited EGF-stimulated invasion. In conclusion, geldanamycin inhibited cancer cell proliferation, down-regulated oncoproteins, and inhibited EGF-induced invasion in thyroid cancer cell lines.

Topics: Adenocarcinoma, Follicular; Adenoma, Oxyphilic; Antibiotics, Antineoplastic; Apoptosis; Benzoquinones; Carcinoma, Papillary; Cell Division; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; HSP90 Heat-Shock Proteins; Humans; In Vitro Techniques; Lactams, Macrocyclic; Mutation; Proto-Oncogene Proteins c-raf; Quinones; Thyroid Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Aminopeptidase N (APN)/CD13 is a transmembrane ectopeptidase expressed on a wide variety of cells. However, the precise function of APN/CD13 in tumor cells and the relationship of APN/CD13 to thyroid cancer remain unclear. In our study, we quantified the expression of APN/CD13 and additionally dipeptidyl peptidase IV (DPIV)/CD26 in thyroid carcinoma cell lines and in tissues of patients with thyroid carcinomas. Undifferentiated anaplastic thyroid carcinomas expressed more APN/CD13 than differentiated thyroid carcinomas. DPIV/CD26 showed an opposite expression pattern. We detected higher levels of DPIV/CD26 in follicular thyroid carcinomas (FTCs) and papillary thyroid carcinomas than in undifferentiated anaplastic thyroid carcinomas. In the undifferentiated thyroid carcinoma cell line 1736, APN/CD13 mRNA expression could be increased by epidermal growth factor, basic fibroblast growth factor, interleukin-6, and tumor necrosis factor alpha. FTC-133 cells stably transfected with an expression vector for APN-enhanced green fluorescent protein showed a higher migration rate than FTC-133 cells transfected with the enhanced green fluorescent protein-control plasmid. Overexpression of APN/CD13 in stably transfected cells is associated with down-regulation of N-myc down-regulated gene (NDRG)-1, melanoma-associated antigen ME491/CD63, and DPIV/CD26 gene expression. Inhibition of APN/CD13 mRNA expression by small interfering RNA induced NDRG-1, ME491/CD63, and DPIV/CD26 mRNA expression in cells of the undifferentiated thyroid carcinoma cell line C643. We conclude that APN/CD13-associated down-regulation of NDRG-1, ME491/CD63, and DPIV/CD26 in thyroid carcinoma cells is an important step of tumor progression to more malignant phenotypes, and we underline the important role of APN/CD13 as mediator in a multimolecular process regulating cell migration.

Topics: Adult; Aged; Carcinoma; CD13 Antigens; Cell Line, Tumor; Cell Movement; Dipeptidyl Peptidase 4; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Male; Middle Aged; Thyroid Neoplasms; Transfection; Tumor Cells, Cultured

Epidermal growth factor (EGF) has widespread growth effects, and in some tissues proliferation is associated with the nuclear localization of EGF and epidermal growth factor receptor (EGFR). In the thyroid, EGF promotes growth but differs from thyrotropin (TSH) in inhibiting rather than stimulating functional parameters. We have therefore studied the occurrence and cellular distribution of EGF and EGFR in normal thyroid, in Graves' disease, where growth is mediated through the thyrotropin receptor (TSHR), and in a variety of human thyroid tumors. In the normal gland the staining was variable, but largely cytoplasmic, for both EGF and EGFR. In Graves' disease there was strong cytoplasmic staining for both EGF and EGFR, with frequent positive nuclei. Nuclear positivity for EGF and particularly for EGFR was also a feature of both follicular adenomas and follicular carcinomas. Interestingly, nuclear staining was almost absent in papillary carcinomas. These findings document for the first time the presence of nuclear EGF and EGFR in thyroid. Their predominant occurrence in tissues with increased growth (Graves' disease, follicular adenoma, and carcinoma) may indicate that nuclear EGF and EGFR play a role in growth regulation in these conditions. The absence of nuclear EGF and EGFR in papillary carcinomas would suggest that the role played by EGF in growth control differs between papillary carcinoma and follicular adenomas/carcinomas of the thyroid.

Topics: Adenoma; Carcinoma; Carcinoma, Papillary; Cell Nucleus; Epidermal Growth Factor; ErbB Receptors; Goiter, Nodular; Graves Disease; Humans; Immunohistochemistry; Reference Values; Thyroid Gland; Thyroid Neoplasms; Tissue Distribution

Cultured human thyroid cells in collagen gel culture were examined on cell morphology and the production of thyroglobulin (Tg), triiodothyronine (T3) and thyroxine (T4) which are components of functional differentiation.. Thyroid cells obtained from normal human thyroid tissues (four cases), follicular adenoma tissues (three cases), papillary carcinoma tissues (three cases), and follicular carcinoma tissue (one case), were cultured in collagen gel. Then these cultured cells were observed on cellular morphology and production of Tg, T3 and T4. Moreover, changes in morphological characteristics and production of Tg, T3 and T4 induced by addition of thyroid stimulating hormone (TSH) and epidermal growth factor (EGF) to medium in collagen gel culture were determined.. Normal and tumor cells in collagen gel culture formed colonies and follicles with Tg production, similar to in vivo-like three-dimensional cellular structures and functions. Normal thyroid cells stimulated TSH induced more Tg and produced morphological changes, i.e. enlarged follicular lumens and increased the height of follicular cells, but did not promote cell proliferation. Reversely, normal thyroid cells stimulated with EGF promoted cell proliferation, but did not change morphological findings and did not increase production of Tg, T3 and T4.. These findings suggest that collagen gel culture is useful for observing the effects of stimulation by cell growth factor on the morphological and functional differentiation of human thyroid cells.

Topics: Cell Differentiation; Cell Division; Collagen; Culture Media; Epidermal Growth Factor; Humans; Thyroglobulin; Thyroid Gland; Thyroid Neoplasms; Thyrotropin; Thyroxine; Triiodothyronine; Tumor Cells, Cultured

Topics: Adenocarcinoma, Follicular; Carcinoma; Cathepsin L; Cathepsins; Cell Differentiation; Cell Division; Colforsin; Cysteine Endopeptidases; Endopeptidases; Enzyme Induction; Enzyme Precursors; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Insulin; Lysosomes; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Tetradecanoylphorbol Acetate; Thyroid Neoplasms; Thyrotropin; Tumor Cells, Cultured

To characterize the influence of six factors on human thyroid tissues at the cell-proliferation level. These six factors were the epidermal growth factor (EGF), the luteinizing-hormone-releasing hormone (LHRH), triiodothyronine, thyroxine, estradiol and gastrin.. Forty-eight human thyroid specimens were obtained from surgical resection and maintained alive for 48 h ex vivo (in vitro) under organotypic culture conditions. These specimens comprised 35 benign cases (17 multinodular goiters and 18 adenomas) and 13 cancers. Cell proliferation in the control and treated conditions (at a 5 nM dose) was assessed by means of the thymidine labeling index, which enables the percentage of cells in the S phase of the cell cycle to be determined in accordance with autoradiographic procedures.. The results show that, of the six factors tested here, EGF significantly (P < 0.05 to P < 0.001) increased cell proliferation in the greatest number of cancers as compared to what happened with the remaining five. Each of these six factors significantly increased or decreased proliferative cell activity in some 10%-30% of the cases under study.. Triiodothyronine, thyroxine, LHRH and gastrin may increase or decrease cell proliferation in human thyroid tissues, whether benign or malignant, to the same extent as other hormones and/or growth factors such as thyrotropin, EGF, insulin-like growth factor 1, transforming growth factor beta1 and estradiol the effects of which on thyroid cell proliferation are already well documented in the literature.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Cell Division; Epidermal Growth Factor; Estradiol; Female; Gastrins; Gonadotropin-Releasing Hormone; Hormones; Humans; Male; Middle Aged; Organ Culture Techniques; Retrospective Studies; Thyroid Diseases; Thyroid Neoplasms; Thyroxine; Triiodothyronine

An imbalance between the activity of matrix metalloproteinases (MMPs) (proteolytic enzymes that degrade protein components of the extracellular matrix) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), may be one of the mechanisms responsible for tumor cell invasion. We have investigated the regulation of MMP-1 and TIMP-1 gene expression in benign and malignant (follicular, anaplastic, and papillary) human thyroid cells. As expected of cells with invasive potential, detectable MMP-1 messenger RNA (mRNA) levels were observed in malignant cells under basal conditions, in contrast to undetectable levels in benign cells. Exposure of these cells, for 1 h, to the active phorbol ester, phorbol 12-myristate 13-acetate (TPA, 100 nmol/L), acting via protein kinase C (PKC), elicited an increase in MMP-1 mRNA, with a peak stimulation after a 3- to 4-h culture period. Epidermal growth factor (EGF, 25 ng/mL), however, acting via protein tyrosine kinase (PTK), stimulated such gene expression in malignant cells but failed to do so in benign cells. TIMP-1 mRNA was not significantly altered by the TPA-PKC, EGF-PTK, or TSH-protein kinase A (PKA) pathways in malignant cells. In benign cells, however, TPA induced a small, though significant, increase in TIMP-1. The MMP-1 stimulation by EGF and lack of TPA-induced rise in TIMP-1 in malignant cells, in sharp contrast to the effects obtained in benign thyrocytes, seems to indicate that the MMP: TIMP balance favors a more extensive extracellular matrix protein breakdown by malignant thyrocytes, as expected of cells exhibiting invasive capacity. TSH (10-500 microU/mL) failed to significantly influence basal MMP-1 or TIMP-1 mRNA levels, but it caused a dose-dependent inhibition in TPA- and EGF-induced MMP-1 mRNA in malignant cells, and TPA-stimulated MMP-1 and TIMP-1 in benign cells. The repressive action of TSH on MMP-1 mRNA was mimicked by forskolin and 8-bromo-cAMP and was abrogated by the PKA inhibitor, H-89, suggesting that the TSH inhibitory action is PKA-mediated. In conclusion, the present study provides novel data on MMP-1 and TIMP-1 gene expression and their modulation by the major signal transduction pathways operating in human thyroid cells. Similar and divergent patterns have emerged in the regulation of such gene expression in benign and malignant human thyrocytes, in many instances in accord with the concept of MMP playing the role of stimulating, and TIMP inhibiting, cell invasion. Although

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenocarcinoma, Follicular; Carcinoma; Carcinoma, Papillary; Cells, Cultured; Colforsin; Collagenases; Cyclic AMP-Dependent Protein Kinases; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Humans; Matrix Metalloproteinase 1; Protein Kinase C; Protein-Tyrosine Kinases; RNA, Messenger; Tetradecanoylphorbol Acetate; Thyroid Neoplasms; Thyrotropin; Tissue Inhibitor of Metalloproteinase-1

Thyroid stimulating hormone (TSH) causes differentiation and epidermal growth factor (EGF) causes dedifferentiation of thyroid cells in vitro. In undifferentiated thyroid cancer cell lines, TSH stimulates tumor cell migration and invasion, a dedifferentiated function, presumably due to an escape of tumor cells from the control of differentiating growth factors. In a highly differentiated thyroid carcinoma cell line of Hürthle cell origin (XTC), we tested the hypothesis that TSH would stimulate thyroglobulin secretion (a differentiated function) more than EGF, and EGF would stimulate invasion (a de-differentiated function) more than TSH. Proliferation, adhesion, cell migration and invasion were measured by the MTT assay, human thyroglobulin by RIA and protease activity by substrate-gel zymography. TSH induced differentiated morphologic changes in XTC cells and stimulated secretion of human thyroglobulin in a dose dependent manner, whereas EGF did not. The effects of TSH on growth, adhesion, migration and invasion were dose dependent and biphasic, with an increase at low and a decrease at high concentrations of TSH. These effects were always more pronounced than those observed with EGE Gelatinolytic activity, consistent with metalloproteinase activity was revealed by zymography, but the pattern of secretion was not altered by neither TSH nor EGF. These results suggest, that TSH has pleiotropic effects on differentiated thyroid cancer cells in vitro that involve differentiated morphology and function but also affect features commonly associated with the malignant in vitro phenotype.

Topics: Cell Adhesion; Cell Differentiation; Cell Division; Cell Movement; Dose-Response Relationship, Drug; Epidermal Growth Factor; Extracellular Matrix Proteins; Gelatin; Humans; Metalloendopeptidases; Neoplasm Invasiveness; Thyroglobulin; Thyroid Neoplasms; Thyrotropin; Tumor Cells, Cultured

Autocrine stimulation of growth is a hallmark of many tumor cell lines. In this work we investigated the synthesis and secretion of growth factors and the expression of their corresponding receptors in HTC-TSHr thyroid carcinoma cells. These cells synthesize epidermal growth factor (EGF) receptors and platelet-derived growth factor beta (PDGF beta) receptors and in addition transforming growth factor alpha (TGF alpha), PDGF-A and PDGF-B chains, respectively. Addition of EGF or PDGF-BB to the culture medium resulted in growth inhibition of HTC-TSHr cells. In contrast, treatment of the cells with low concentrations of neutralizing anti-TGF alpha antibodies or tyrosine kinase inhibitors led to stimulation of cell proliferation. Low concentrations of neutralizing anti-PDGF-B antibodies did not affect growth of the cells. As expected, cell proliferation was inhibited when high concentrations of either neutralizing anti-TGF alpha antibodies or anti-PDGF-B antibodies were applied. PDGF-AA did not influence growth of HTC-TSHr cells. We conclude that growth of HTC-TSHr thyroid carcinoma cells is influenced by two autocrine loops between TGF alpha and EGF receptors and between PDGF-B and PDGF beta receptors. However, our data suggest that excessive activation of tyrosine kinase receptors in these cells results in a relative inhibition rather than stimulation of growth.

Topics: Cell Division; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Molecular Weight; Platelet-Derived Growth Factor; Protein-Tyrosine Kinases; Receptors, Platelet-Derived Growth Factor; Thyroid Neoplasms; Transforming Growth Factor alpha; Tumor Cells, Cultured

We previously established transplantable rat thyroid carcinoma cell lines in vivo from primary thyroid tumors induced by N-bis-(2-hydroxypropyl)nitrosamine (DHPN). In the present study, an insulin-like growth factor I (IGF-I)-responsive cell line (TRTC-G1-C-A4) in culture was derived from one (well differentiated papillary type) of these carcinoma cell lines G1. TRTC-G1-C-A4 cells were found to exhibit specific saturable binding of IGF-I with a Kd of 1.16 nM at approximately 43.6 fmol/10(5) cells. Inclusion of IGF-I (10 and 50 ng/ml) in the culture medium resulted in a significant increase of [3H]thymidine incorporation and marked cell proliferation. IGF-II (10 ng/ml) and insulin (1 microgram) produced no such effects. The molecular weight of IGF-I receptors on the cell membrane was determined by Western blotting analysis, a single band of binding proteins with a molecular weight of 125 kDa being evident under non-reducing conditions. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that the TRTC-G1-C-A4 cells contained IGF-I receptor mRNA with a sequence corresponding to that determined from rat uterus. These results demonstrate that the IGF-I receptor can be expressed in a thyroid carcinoma with an important contribution to cell growth.

Topics: Animals; Blotting, Western; DNA, Neoplasm; Epidermal Growth Factor; Insulin-Like Growth Factor I; Rats; Receptor, IGF Type 1; RNA, Messenger; Thymidine; Thyroid Neoplasms; Thyrotropin; Tumor Cells, Cultured

IGFs and IGF binding proteins (IGFBPs) appear to serve as regulators of non-malignant thyroid cells from several species, but little is known about their role in thyroid malignancy. We have examined IGFBP production and hormonal regulation in two human thyroid follicular carcinoma cell lines; FTC-133 line derived from a local tumor recurrence and FTC-236 cells from a tumor metastasis. Under basal conditions these cell lines produced IGFBP-3, IGFBP-4 and IGFBP-2. In both cell lines, EGF or TPA stimulated IGFBP-3 production while TSH or forskolin inhibited IGFBP-3 production and reduced the stimulation of IGFBP-3 seen with EGF or TPA. IGFBP-4 production was increased in the presence of TSH, forskolin, and EGF and was reduced by TPA. mRNA assessment revealed that IGFBP-3 mRNA, more abundant in FTC-236 than FTC-133 cells, increased in the presence of EGF or TPA, while IGFBP-4 mRNA content was increased in the presence of TSH, EGF, and forskolin. These results indicate that IGFBP production in human thyroid follicular carcinoma clones is under specific hormonal regulation. IGFBP-3 production is increased by dedifferentiation factors such as EGF and TPA and inhibited by TSH and forskolin, which enhance differentiated function. The highly specific regulation of IGFBP-3 and IGFBP-4 suggests a potential role for these peptides in modulating malignant thyroid growth.

Topics: Adenocarcinoma, Follicular; Blotting, Northern; Carrier Proteins; Colforsin; Endopeptidases; Epidermal Growth Factor; Humans; Immunosorbent Techniques; Insulin-Like Growth Factor Binding Protein 2; Insulin-Like Growth Factor Binding Protein 4; Insulin-Like Growth Factor Binding Proteins; RNA, Messenger; Tetradecanoylphorbol Acetate; Thyroid Neoplasms; Thyrotropin; Tumor Cells, Cultured

We have shown recently that epidermal growth factor (EGF) enhanced invasion and growth of differentiated thyroid cancer cells in vitro and in vivo. The present study analyzed the effects of transforming growth factor alpha (TGF-alpha) on invasion and growth of a follicular thyroid cancer cell line (FTC133) and whether blocking the EGF receptor by a monoclonal antibody (Mab528) or blocking the tyrosine kinase of the receptor by genistein abolished the EGF- and TFG-alpha-mediated effects. Growth and invasion (penetration of 8-microns pore polycarbonate membranes coated with Matrigel) were determined by the dimethylthiazol-diphenyltetrazolium bromide assay. Epidermal growth factor (10 ng/l) stimulated invasion of FTC133 by 42% and TGF-alpha (10 ng/l) stimulated invasion of FTC133 by 27% (p < 0.02). Both growth factors also enhanced growth by 62% (EGF) and 30% (TGF-alpha) (p < 0.003). Epidermal growth factor receptor antibodies (1 microgram/ml) abolished EGF-mediated stimulation of invasion and growth completely and that of TGF-alpha by 93%. At 100 ng/ml genistein reversed EGF and TGF-alpha stimulation, and at 1 microgram/ml it inhibited invasion (27%) and growth (40%) of unstimulated FTC133 (p < 0.02). We conclude that TGF-alpha stimulates invasion and growth of follicular thyroid cancer by binding to the EGF receptors, that EGF- and TGF-alpha-mediated effects can be blocked by antagonism to the EGF receptor and to tyrosine kinase, and that genistein not only neutralized EGF and TGF-alpha effects but also inhibited invasion and growth of unstimulated FTC133.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antibodies, Monoclonal; Cell Division; Cell Movement; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Female; Genistein; Humans; Isoflavones; Protein-Tyrosine Kinases; Thyroid Neoplasms; Transforming Growth Factor alpha; Tumor Cells, Cultured

Aggressiveness of follicular (FTC) and papillary thyroid cancer (PTC) varies widely. Tumorigenesis is associated with an imbalance of growth-promoting and growth-constraining factors. We investigated the effects of thyrotropin (TSH), epidermal growth factor (EGF) and transforming growth factor beta 1 (TGF-beta 1) on invasion and growth of 3 FTC- and 2 PTC-cell lines. Invasion (penetration through an 8 microns pore membrane, covered by Matrigel) and growth were measured using the MTT-method. EGF (10 ng/ml) and TSH in low concentrations (1 mU/ml) stimulated invasion and growth of FTC and PTC, whereas TGF-beta 1 (10 ng/ml) and TSH in high concentrations (100 mU/ml) were inhibiting. The parental cell line FTC133 was considerably more responsive to all growth factors than the metastatic clones. Invasion of FTC133 was enhanced by 42% (EGF) and 21% (TSH), invasion of FTC236 by 8% (EGF and TSH). Conversely, invasion of FTC133 was inhibited by 32% (TGF-beta 1) and 21% (TSH), invasion of FTC236 by 18% (TGF-beta 1) and 11% (TSH). TSH, EGF and TGF-beta 1 have an important impact on differentiated thyroid cancer cells and metastases may have developed by escaping from the normal control of growth factors.

Topics: Adenocarcinoma, Follicular; Animals; Carcinoma, Papillary; Cell Division; Cell Line; Epidermal Growth Factor; Growth Substances; Humans; Lymph Nodes; Lymphatic Metastasis; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Thyroid Gland; Thyroid Neoplasms; Thyroidectomy; Thyrotropin; Transforming Growth Factor beta; Tumor Cells, Cultured

Antiproliferative effects of somatostatin (SRIH) analogs were investigated in human thyroid carcinoma cell lines. Membrane preparations from six human thyroid cell lines, including follicular (RO 87-M-1 and RO 82-W-1), papillary (NPA87), and anaplastic (RO 90-D-1) carcinomas, a follicular adenoma, as well as benign and malignant human thyroid tissues, contained high affinity SRIH-binding sites. Ligand-binding studies ([125I]Tyr11-SRIH) demonstrated mean dissociation constants ranging from 114-224 pmol/L, and 20-154 fmol/mg membrane protein mean receptor sites, in cell lines. Four cell lines were grown for 3 days in monolayers with SRIH analogs (octreotide or MK0678) at concentrations from 0.05-100 nmol/L in serum-free medium to assess changes in cell numbers. At the highest dose, MK0678 produced dose-dependent inhibition of growth in RO 87-M-1 and NPA87, each to about 70% of that in control cells. Octreotide produced dose-dependent stimulation of growth in RO 87-M-1 cells, but caused growth inhibition in NPA87 cells, with loss of effect at the highest dose. RO 82-W-1 did not respond to MK0678, yet caused biphasic inhibition with octreotide (75% and 45% of control cell numbers at 0.05 and 100 nmol/L doses, respectively). Anaplastic cells, RO 90-D-1, did not respond to either analog despite similar ligand binding. Addition of epidermal growth factor (100 micrograms/L) or TSH (200 mU/L) increased the sensitivity of RO 87-M-1 cells to growth inhibition by the lowest dose of MK0678, producing biphasic dose-response curves. In conclusion, the present data demonstrate specific SRIH binding to membranes of thyroid carcinoma cells and tissues as well as discordant growth effects of different SRIH analogs on the same cell lines. This may be a result of differential stimulation and regulation of distinct SRIH receptor subtypes.

Topics: Carcinoma; Cell Division; Epidermal Growth Factor; Humans; Receptors, Somatotropin; Somatostatin; Thyroid Neoplasms; Thyrotropin; Tumor Cells, Cultured

The aggressiveness of follicular thyroid cancer (FTC) varies widely, and metastasis is the primary cause of death. Uncontrolled proliferation of cancer cells may be associated with loss of growth factor control. We investigated the effects of stimulating (epidermal growth factor [EGF]; thyreotropin [TSH] in low concentrations) and inhibiting growth factors (transforming growth factor beta 1 [TGF beta 1]; TSH in high concentrations) on invasion and growth of FTC cell lines from the thyroid tumor (FTC133) and from the lymph node (FTC236) and lung (FTC238) metastases of the same patient. Invasion-penetration through an 8 microns pore membrane, covered by Matrigel (basement membrane)-and growth were measured using the MTT-method. EGF (10 ng/ml) and TSH in low concentrations (1 mU/ml) stimulated invasion and growth of all FTC cell lines, but the amplitude of stimulation differed significantly. The parental cell line FTC133 was considerably more responsive to growth factor stimulation than the metastatic clones. Invasion of FTC133 was enhanced by 42% (EGF; p < 0.02) and 21% (TSH; p < 0.01), invasion of FTC236 by 8% (EGF; p < 0.02) and 8% (TSH; p < 0.01), and invasion of FTC238 by 9% (EGF; p < 0.02) and 8% (TSH; p < 0.01). Conversely, invasion and growth of FTC133 were significantly more inhibited by TGF beta 1 (10 ng/ml) and supraphysiologic concentrations of TSH (100 mU/ml) than the cell lines from the lymph node and lung metastases. At day 7, invasion of FTC133 was inhibited by 32% (TGF beta 1; p < 0.02) and 21% (TSH; p < 0.01), invasion of FTC236 by 18% (TGF beta 1; p < 0.02) and 11% (TSH; p < 0.01), and invasion of FTC238 by 16% (TGF beta 1; p < 0.02) and 12% (TSH; p < 0.01). Moreover, we analyzed growth factor independence in minimally supplemented or unsupplemented medium. Growth, but no invasion was evident when cells were cultured completely unsupplemented over 7 days. These results suggest that metastatic FTCs may have developed by escaping from the normal control of TSH and other growth factors.

Topics: Adenocarcinoma, Follicular; Cell Division; Culture Media; Epidermal Growth Factor; Growth Substances; Humans; In Vitro Techniques; Neoplasm Invasiveness; Neoplasm Metastasis; Thyroid Neoplasms; Thyrotropin; Transforming Growth Factor beta; Tumor Cells, Cultured

The prognosis of patients with follicular (FTC) and papillary (PTC) thyroid cancer depends on age and the size and extent of the tumor. Differentiated thyroid cancers bind more epidermal growth factor (EGF) than normal thyroid tissue, but the role of EGF in the proliferation and invasion of thyroid cancer is unknown. We investigated the effects of EGF on growth, migration, and invasion in a follicular thyroid cancer that metastasized to cervical lymph nodes and the lung (FTC 133, primary; FTC 236, lymph node; and FTC 238, lung metastasis) and in a papillary thyroid cancer (PTC-UC3). As measured by the formazan method (dimethylthiazol-diphenyltetrazolium bromide), EGF caused a dose- and time-dependent increase in the growth of FTC 133 and PTC-UC3 by 25%, but its stimulatory effect on growth of the metastatic FTC subclones was smaller (FTC 236, 14%; FTC 238, 8%; P < 0.001). EGF also enhanced the ability of all cell lines to migrate (through 8-microns pore membranes without Matrigel) or invade (membranes with Matrigel). Migration of FTC 133 was enhanced from 86% migrated tumor cells to 95% after 72 h (P < 0.02). Again, stimulation by EGF was lower in FTC 236 and FTC 238. EGF increased migration in PTC-UC3 from 49% to 58%. EGF stimulated invasion of FTC 133 from 17.5% to 24.9%. In the absence of EGF, FTC 238 was the most invasive tumor, but, again, the EGF stimulatory effect was less pronounced than in the primary tumor. EGF stimulated the invasion of PTC-UC3 from 10.9% to 14.3% (P < 0.03). EGF also stimulated the growth of thyroid cancer xenografts in nude mice. Although all FTC cell lines were 100% tumorigenic in nude mice, PTC-UC3 was less tumorigenic. However, after sc inoculation of EGF-pretreated tumor cells, 7 of 10 animals developed tumors (mean size, 2.3 cm3) compared to 2 of 10 animals (mean size, 1.4 cm3) in the control group (P < 0.02). In summary, EGF stimulates the growth and invasion of differentiated thyroid cancer cells in culture and in nude mice. Escape from growth factor control, such as in FTC 236 and FTC 238, may be an important step in the development of metastatic thyroid cancer.

Topics: Adenocarcinoma, Follicular; Animals; Carcinoma, Papillary; Cell Division; Cell Movement; Epidermal Growth Factor; Humans; Lung Neoplasms; Lymphatic Metastasis; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Thyroid Neoplasms; Tumor Cells, Cultured

Invasion and metastasis may be caused by the escape of tumor cells from the negative control of growth factors. We analyzed the effects of transforming growth factor-beta 1 (TGF beta 1) on growth, migration, invasion, and adhesion in three follicular thyroid cancer cell lines (FTC133, primary; FTC236, lymph node metastasis; FTC238, lung metastasis) from one patient and in a papillary line (PTC-UC3). Cell growth was measured by dimethylthiazol-diphenyltetrazolium bromide assays, and migration (basal or epidermal growth factor stimulated) was determined by the ability of cells to penetrate 8-microns pore membranes that were covered with Matrigel for invasion assays. Moreover, we studied tumor cell adhesion to collagen type IV, fibronectin, and laminin. TGF beta 1 inhibited growth in FTC (FTC133, by 31%; FTC236, 15%; FTC238, 17%; P < 0.008), but not in PTC. Migration was inhibited in all cell lines. TGF beta 1 inhibited epidermal growth factor-stimulated migration of FTC133 by 43% vs. 29% without epidermal growth factor (P < 0.03). TGF beta 1 also inhibited invasion (FTC133, 32%; FTC236, 18%; FTC238, 16%; PTC-UC3, 32%; P < 0.02). All cell lines adhered preferably to collagen type IV and fibronectin. TGF beta 1 enhanced adhesion. Again, these effects were less pronounced in the FTC metastases. In conclusion, TGF beta 1 inhibits the growth, migration, and invasion of thyroid cancer cells in vitro. It enhances adhesion to components of the extracellular matrix. Metastatic thyroid tumors may be less responsive to the negative regulation of TGF beta 1.

Topics: Adenocarcinoma, Follicular; Carcinoma, Papillary; Cell Adhesion; Cell Division; Cell Movement; Epidermal Growth Factor; Humans; Neoplasm Invasiveness; Thyroid Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured

The signal transduction of TSH in invasion and growth of FTC 133, a human follicular thyroid cancer cell line, was investigated. TSH (0.01-1 mIU/ml) stimulated invasion of FTC 133 by 21% and growth by 20% of basal. Cyclic AMP-stimulators and inhibitors had no effect at any concentration. The PKC-agonist TPA enhanced invasion and growth by 15%, whereas staurosporine, a PKC-antagonist, inhibited them by 32% and 60%, respectively. The latter also reversed TSH stimulation. EGF enhanced invasion (42%) and growth of FTC 133 (25%). Staurosporine did not reverse EGF stimulation. The tyrosine kinase antagonist genistein reversed EGF, but not TSH stimulation. Pertussis toxin inhibited invasion (18%) and growth (22%). Cholera toxin was less inhibitive. We demonstrated for the first time, that TSH stimulates invasion and growth of human thyroid cancer cells in vitro by PKC- rather than PKA-stimulation.

Topics: Alkaloids; Cell Division; Cholera Toxin; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Activation; Epidermal Growth Factor; Humans; Neoplasm Invasiveness; Pertussis Toxin; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate; Thyroid Neoplasms; Thyrotropin; Tumor Cells, Cultured; Virulence Factors, Bordetella

Serum levels of epidermal growth factor (EGF) were investigated in 31 patients with differentiated carcinoma of thyroid. Patients with carcinoma had significantly decreased basal levels of serum EGF, however this decrease in serum EGF occurred only in the group of patients which had been evaluated at an interval of six weeks after ablation of residues of normal thyroid with radioiodine. In patients that had been treated with radioiodine for cancer this decrease was not observed. The change in serum EGF was not dependent on thyroidal function and did not correlate with the serum thyreoglobulin level. It is presumed that decreased serum EGF is a consequence of functional changes of platelets after whole body irradiation at ablative dose of radioiodine.

Topics: Adult; Aged; Cell Differentiation; Epidermal Growth Factor; Female; Humans; Iodine; Iodine Radioisotopes; Male; Middle Aged; Platelet Count; Radioimmunoassay; Sensitivity and Specificity; Thyroglobulin; Thyroid Neoplasms; Thyroxine

Apparently complex modulatory effects of alpha-interferon (alpha-IFN), tumor necrosis factor (TNF), and epidermal growth factor (EGF) have been found in neoplastic human thyroid cells, which could possibly affect the final outcome in neoplastic disease. This was achieved by examining the influence of alpha-IFN, TNF, and EGF alone and in combination, on human leukocyte antigen-DR (DR) antigen expression and viability of neoplastic and non-neoplastic human thyroid cells in culture. alpha-IFN-induced DR antigen expression on non-neoplastic human thyroid cells, whereas TNF-alpha or EGF alone were ineffective. The addition of the same TNF-alpha concentrations (10 to 100 ng/ml) to alpha-IFN enhanced the expression of DR antigens compared with the effect of alpha-IFN alone. However, EGF inhibited alpha-IFN-induced DR on the same cells and at the same concentrations (10 to 500 ng/ml) at which the growth factor alone was ineffective. In contrast to the common pattern of cytokine effects on DR expression of all nonmalignant thyroid cell lines, neoplastic thyroid cell lines showed divergent responses to alpha-IFN, TNF-alpha, and EGF. In three malignant thyroid cell lines that were DR negative (follicular carcinoma WRO 82-1 and NRO 87-1 cell lines, and anaplastic carcinoma ARO 81-1), DR antigen could be induced by alpha-IFN and enhanced by TNF-alpha, whereas EGF was ineffective. In a fourth cell line (an anaplastic carcinoma SW1736) alpha-IFN, TNF-alpha, and EGF alone were capable of inducing DR, and a combination of either TNF-alpha and EGF with alpha-IFN potentiated DR induction. In a fifth neoplastic cell line (papillary carcinoma, NPA) that constitutively expressed surface DR, its expression was inhibited by both alpha-IFN and TNF-alpha and was not affected by EGF.

Topics: Antibodies; Antigens, Neoplasm; Cell Survival; Cells, Cultured; Chromium Radioisotopes; Cytokines; Epidermal Growth Factor; HLA-DR Antigens; Humans; Interferon-alpha; Iodine Radioisotopes; Kinetics; Stimulation, Chemical; Thyroid Gland; Thyroid Neoplasms; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The epidermal-growth-factor receptor (EGF-r) and its ligands are involved in the control of proliferation of both normal and neoplastic thyroid epithelium. Autocrine stimulation of growth involving this receptor system has been identified in several types of human neoplasia and we were interested to determine whether it might occur in human thyroid tumours. We have therefore examined an archival series of thyroid tumours and non-neoplastic pathologies for expression of the EGF-r and 2 of its ligands, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF), using immunohistochemistry. We found evidence of expression of both the EGF-r and the TGF-alpha in the majority of thyroid tumours, with a trend to higher expression in more malignant neoplasms. We also found variable levels of expression of EGF-r and TGF-alpha in all cases of thyroiditis examined. We conclude that there is a potential autocrine loop involving the EGF-r system in both neoplastic and non-neoplastic conditions of the human thyroid.

Topics: Adenocarcinoma; Carcinoma; Carcinoma, Papillary; Epidermal Growth Factor; ErbB Receptors; Humans; Retrospective Studies; Thyroid Gland; Thyroid Neoplasms; Thyroiditis; Transforming Growth Factor alpha

The expression of epidermal growth factor (EGF) and c-myc oncogene product was studied immunohistochemically in 65 benign and malignant human thyroids. Normal thyroid tissues were usually negative with each antibody. Benign and malignant neoplastic thyroid tissues demonstrated a high percentage of stained cells. However, there was no significant difference in positivity between benign and malignant neoplastic tissues. In Graves' disease 25% of the cases were positive for EGF, and 100% for c-myc product. The effect of EGF or c-myc expression on prognosis was also examined in 42 patients with papillary thyroid carcinoma. When the cases were divided into two groups with regard to their EGF staining score (highly-expressed and poorly-expressed groups), the frequency of highly-expressed tumours was significantly higher in the recurrence-positive group compared with the recurrence-free group (86% vs 57%), but there was no significant difference between the groups in respect of c-myc expression.

Topics: Blotting, Western; Epidermal Growth Factor; Follow-Up Studies; Humans; Immunohistochemistry; Prognosis; Proto-Oncogene Proteins c-myc; Thyroid Gland; Thyroid Neoplasms

The total contribution of the thyroid to epidermal growth factor (EGF) levels in blood and urine was determined in 18 patients cured from thyroid cancer at the time of thyroxine treatment and 6 weeks off it in the state of athyroidism. As compared to the euthyroid state, the athyrodism was associated with a drop in the urinary EGF excretion from 24.8 +/- 8.4 to 11.8 +/- 4.6 ng/mg creatinine (p less than 0.0001) and some increase in the EGF level in blood serum from 159.5 +/- 55.0 to 191.2 +/- 41.0 pM (p = 0.023). We conclude that the thyroid affects the synthesis and/or blood delivery of EGF and is responsible for approximately one half of its urinary excretion.

Topics: Adult; Epidermal Growth Factor; Female; Humans; Hypothyroidism; Male; Middle Aged; Thyroid Neoplasms; Thyroxine

The content of epidermal-growth-factor receptor (EGFr) and its relation to TSH-response were examined in 27 malignant thyroid tumors (5 follicular, 6 papillary, 5 medullary, 11 anaplastic carcinomas) and in 30 tumor-like lesions (21 hyperplastic goiters and 9 toxic adenomatous goiters). Normal 12 thyroid tissues adjacent to benign tumors with no evidence of macroscopic or microscopic abnormalities were used as control. All thyroid plasma membranes tested showed specific EGF binding. In membranes from toxic adenomas (2.18 +/- 0.73 fmoles/mg protein) and papillary carcinomas (2.80 +/- 0.80) the EGF binding was similar to that of normal thyroid membranes (2.32 +/- 0.73). Hyperplastic goiters showed an EGF binding (4.4 +/- 0.82) slightly higher than normal tissue. The highest and the lowest EGF binding values were found in anaplastic (11.8 +/- 2.78) and medullary (0.50 +/- 0.39) carcinomas, respectively. An inverse correlation between EGFr content and TSH-response was found when anaplastic thyroid tumors were compared to tumor-like lesions. However no correlation was observed in medullary carcinomas which also failed to respond to TSH and in papillary carcinomas which partially respond to thyrotropin.

Topics: Cell Membrane; Cyclic AMP; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Phospholipids; Thyroid Diseases; Thyroid Gland; Thyroid Neoplasms; Thyrotropin

Our studies using thyrocyte membranes from different human thyroid tissues, monolayer cultures of human thyrocytes, and the permanant cell line FTC-133 demonstrate the stimulatory effect of TSH on metabolism, DNA synthesis, and cell growth in human thyrocytes. Up- and down-regulation of cAMP cell content fails to show direct effects on DNA synthesis and cell growth in primary thyrocyte cultures in man. Increased AC responsiveness to TSH in adenomatous human thyroid tissues, when compared to normal thyroids of the same patient (p less than 0.005), is thus of only questionable importance for thyroid tumor growth. The permanant cell line FTC-133 was established from differentiated follicular human thyroid cancer cells. FTC-133 cells proved to be of particular usefulness in assessing growth regulation of human thyroid tissue. These cells could be propagated in serum free medium, showed thyroglobulin immunoreactivity and EGF receptors, lacked any fibroblast contamination, and responded to TSH and local active growth factors such as EGF and IGF with a stimulated [3H]thymidine incorporation. The latter could be shown in primary cell cultures of normal and pathological human thyrocytes as well. Additional to the stimulatory effect of TSH and IGF on [3H]thymidine incorporation, these substances show an additive effect when incubated simultaneously. Locally active growth factors and endocrine growth stimulation by TSH therefore act synergistically on thyrocyte growth in human thyrocyte cultures. Whether the TSH effect on cell growth is related to its stimulation of AC remains as yet questionable.

Topics: Adenylyl Cyclases; Bucladesine; Cell Division; DNA; Epidermal Growth Factor; Humans; Thyroid Gland; Thyroid Neoplasms; Thyrotropin

Dedifferentiation of human thyroid tumors is frequently found in humans. The effect of retinoids (13 cis-RA) was studied on the proliferation and differentiation of a human follicular cell line in vitro (UCLA R0 82 W-1). A significant and dose-dependent reduction (P less than 0.001) in cell number and [3H] thymidine uptake was found in cells exposed to 13 cis-RA up to 10 microM. Higher concentrations of 13 cis-RA, however, led to a dose-dependent restoration of cell proliferation. Various parameters of differentiation increased under the influence of 13 cis-RA (10 microM) over nonexposed cells. The 125I uptake increased 4-fold over that in control nonexposed cells (P less than 0.05). [125I] Epidermal growth factor binding increased 5-fold, and [125I] human TSH binding increased significantly after exposure to 13 cis-RA (P less than 0.02). Deiodinase activity, however, was significantly lower in 13 cis-RA exposed cells than in control cells. The present study shows that 13 cis-RA (10 microM) drives the tumor cells toward a more normal state of proliferation and differentiation.

Topics: Adenocarcinoma; Binding Sites; Cell Cycle; Cell Differentiation; Cell Survival; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Iodide Peroxidase; Iodine; Thymidine; Thyroid Neoplasms; Thyrotropin; Tretinoin; Tumor Cells, Cultured

We analyzed epidermal growth factor receptors (EGF-R) and the growth stimulatory effects of epidermal growth factor (EGF) in the presence or absence of TSH on cultured human non-neoplastic and Graves' thyroid cells. All cells studied possessed EGF-R composed of two components. There was no significant differences in the binding characters of EGF-R among non-neoplastic thyroid cells whether they were obtained from thyroid tissues adjacent to malignant carcinoma or benign adenoma. Ten nM of EGF stimulated (3H)-thymidine (dTR) incorporation of non-neoplastic thyroid cells by about 50%. However, TSH had no effect on the growth of these cells. Both EGF-R binding parameters and cell proliferation effects of EGF and TSH were simultaneously examined in non-neoplastic thyroid cells from 8 patients. A significant inverse correlation (r = -0.757) was observed between binding affinity (Ka1) and EGF-induced increase of dTR incorporation. Binding capacity (Cmax) did not correlate significantly with dTR incorporation. In Graves' thyroid cells, all parameters of EGF-R were significantly lower than those of non-neoplastic thyroid cells, higher basal dTR incorporation was observed, and their goiter size significantly correlated with EGF-induced increase of dTR incorporation (r = 0.879) and also appeared to correlate inversely with Ka1. These data indicate a close relationship between the binding affinity of EGF-R and thyroid cell growth.

Topics: Cell Division; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Graves Disease; Humans; Thymidine; Thyroid Gland; Thyroid Neoplasms

Epidermal growth factor (EGF) receptors on primary-cultured human thyroid cells from 27 neoplasias (nine adenomas and 18 differentiated carcinomas) were analyzed and compared with those on the cultured nonneoplastic part of human thyroid cells. Total binding of 125I-EGF to the nonneoplastic part, adenoma, and carcinoma cells did not differ significantly. Scatchard analysis showed that the neoplastic human thyroid cells, like their adjacent nonneoplastic counterparts, consistently possessed EGF receptors with two components. In a paired study of five patients, the association constant of the carcinoma cells' high-affinity component (Ka1) was found to be significantly lower than that of adjacent nonneoplastic thyroid cells (P less than 0.05). Furthermore, a study of the cells from 18 carcinomas revealed that overall their Ka1s (4.15 +/- 0.82 x 10(9) M-1, mean +/- SEM) were significantly lower than those of adenoma cells (10.34 +/- 1.51 x 10(9) M-1, n = 9) and of nonneoplastic cells adjacent to them (8.32 +/- 0.84 x 10(9) M-1, n = 23). The difference in Ka1s for adenoma and nonneoplastic thyroid cells was not statistically significant. The number of receptor sites (Cmax) per cell was not significantly different in any of the three. Incorporation of [3H]thymidine (dThd) increased significantly in all kinds of thyroid cells examined following the addition of 10 nM EGF, and the paired study showed that the size of this increase was not significantly different in neoplastic and adjacent nonneoplastic cells. The addition of 300 microunits/ml of thyroid-stimulating hormone caused a significant increase in dThd incorporation by adenoma cells but not by carcinoma or nonneoplastic cells. Furthermore, combined treatment with EGF and thyroid-stimulating hormone additively promoted adenoma cell growth only. A close inverse relationship was observed between Ka1 and the stimulatory effect of EGF on the dThd uptake in both nonneoplastic thyroid cells and adenoma cells. Carcinoma cells also showed similar profiles, but Ka1 relative to dThd increases were much smaller than the other two.

Topics: Adenoma; Carcinoma; Cell Division; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Thyroid Neoplasms; Thyrotropin; Tumor Cells, Cultured

The nature of epidermal growth factor (EGF) receptors in normal and pathological thyroid membranes was studied on a crude membrane fraction (10,000 X g pellet) of tissue homogenate. Optimal binding of 125I-EGF to human thyroid membranes was obtained at 25 degrees C with 1-h incubation at pH 7.4. The study revealed the presence of both high and low affinity receptors in normal and pathological thyroid membranes. The association constants of high affinity receptor (3.2 X 10(-9) mol/l) and of low affinity receptor (1.8 X 10(-8) mol/l) observed in normal thyroid membranes were similar to those of thyroid membranes from neoplastic as well as thyrotoxic thyroid tissues. [3H]thymidine incorporation into DNA of cultured human thyroid cells was stimulated by EGF in a dose-dependent manner. A half-maximal stimulation was found around 1 X 10(-10) mol/l. These results suggest that EGF may have a possible role in the regulation of thyroid growth and function in physiological and pathological situations.

Topics: Animals; Cell Division; Cell Membrane; Cells, Cultured; DNA; Epidermal Growth Factor; ErbB Receptors; Male; Mice; Thyroid Gland; Thyroid Neoplasms; Thyrotoxicosis

Human thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture conditions on cell growth were measured by changes in cell numbers and by stimulation of [3H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of insulin (10 micrograms/ml), transferrin (5 micrograms/ml), hydrocortisone (10 micrograms/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine (10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present at 10(-9) M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system provides a means to study the hormonal control of growth and differentiation in human thyroid cells.

Topics: Cell Differentiation; Cells, Cultured; Culture Media; DNA Replication; Epidermal Growth Factor; Epithelium; Fluorescent Antibody Technique; Hormones; Humans; Thyroglobulin; Thyroid Gland; Thyroid Neoplasms; Thyrotropin; Transferrin