Laminin receptors in differentiated thyroid tumors: restricted expression of the 67-kilodalton laminin receptor in follicular carcinoma cells.

The expression of integrin laminin receptors was investigated in normal thyroid primary cultures; immortalized normal thyroid cells (TAD-2); papillary (NPA), follicular (WRO), and anaplastic (ARO) thyroid tumor cell lines; seven thyroid tumors (four papillary and three follicular carcinomas); and normal thyroid glands. The expression of alpha1beta1, alpha2beta1, alpha3beta1, alpha6beta1, and alpha6beta4 was found in all tumor specimens and in tumor cell lines, whereas normal thyroid cells and TAD-2 cells lacked the expression of alpha6beta4. Despite the presence of several integrin laminin receptors, adhesion of TAD-2, NPA, and ARO cells to immobilized laminin-1 was poor, whereas WRO cells and follicular carcinoma-derived cells displayed a strong adhesion. Indeed, WRO and follicular carcinoma-derived cells showed expression of a nonintegrin laminin receptor, the 67-kDa high affinity laminin receptor (67LR). TAD-2, NPA, and ARO cells as well as nodular goiter, toxic adenoma, follicular adenoma, and papillary carcinoma-derived cells did not express the 67LR. Adhesion of WRO and follicular carcinoma-derived cells to laminin-1 was specifically inhibited by a recombinant polypeptide containing laminin-binding domains of 67LR, demonstrating that this receptor confers to follicular carcinoma cells attachment capacity to laminin. Moreover, tissue specimens from follicular carcinomas expressed the 67LR, whereas follicular adenomas and normal thyroid tissues were negative. In thyroid tumors, integrin receptors, although abundant, participate weakly in adhesion to laminin. The expression in follicular carcinoma cells of a functional, high affinity 67LR together with nonfunctional integrin LM receptors could be responsible for the tendency of follicular carcinoma cells to metastasize by mediating stable contacts with basal membranes.     

Differential expression of IgG Fc binding protein (FcgammaBP) in human normal thyroid tissue,thyroid adenomas and thyroid carcinomas

The genetic events involved in thyroid carcinogenesis are still incompletely understood. Several rearrangements and mutations of oncogenes have been implicated in the development of thyroid papillary carcinomas, follicular adenomas and carcinomas. However, none of these molecular alterations is suitable either as a general marker for the diagnosis of thyroid carcinomas or to differentiate between thyroid follicular adenomas and carcinomas. In order to identify new genes with altered expression which could serve as such markers, we analyzed RNA from thyroid tumor and normal tissue using a novel technique called restriction-mediated differential display. Several differentially expressed genes were identified, including the gene for IgG Fc binding protein (FcgammaBP). Differential expression of FcgammaBP was confirmed by quantitative real-time RT-PCR. Our experiments showed that IgG Fc binding protein (FcgammaBP) is differentially expressed in normal thyroid tissue, thyroid adenomas and thyroid carcinomas. While the FcgammaBP gene is constitutively expressed in normal thyroid tissue, its expression is significantly increased in follicular thyroid adenomas and significantly decreased in papillary and follicular thyroid carcinomas. Thus, measurement of the expression levels of FcgammaBP in thyroid biopsies might help to make the otherwise difficult distinction between a thyroid follicular adenoma and a follicular carcinoma.     

Fas ligand expression in thyroid carcinomas: a potential mechanism of immune evasion.

Fas ligand (FasL) induces apoptosis by cross-linking the Fas receptor and is expressed by cells of the immune system. Recently, FasL was found in malignant tumors, suggesting that it helps them escape immune surveillance by eliminating infiltrating lymphocytes. We investigated the presence of FasL immunohistochemically in 48 thyroid carcinomas and by Western blotting and RT-PCR in 5 thyroid carcinoma cell lines. We found that in contrast to normal thyroid tissue, FasL was highly expressed in all papillary, follicular, and Huerthle carcinomas. Medullary carcinomas lacked or had minimal FasL expression. In papillary carcinomas, high levels of expression correlated independently with aggressive histology and unfavorable clinical presentation. FasL was also present in all thyroid cell lines. Thyroid carcinoma cells killed Fas-sensitive targets in a FasL-dependent manner in a coculture experiment. Cross-linking of Fas induced apoptosis in thyroid carcinoma cells only in the presence of cycloheximide. We conclude that FasL is specifically expressed in thyroid carcinomas of follicular epithelial origin, may help them evade the immune system, and may have prognostic implications in papillary carcinoma, as it is associated with a more aggressive phenotype. Thyroid carcinoma cells avoid Fas-mediated suicide possibly by expressing an inhibitor of the Fas apoptotic pathway.     

Three-dimensional structure of the micro-blood vessels in thyroid tumors analyzed by immunohistochemistry coupled with image analysis.

The structure of micro-blood vessels, one of the most important factors influencing the tumor growth and tumor metastasis among histological types of thyroid malignancy, was analyzed immunochemically by staining tissues for platelet endothelial cell adhesion molecule-1 (PECAM-1). Human thyroid tumor tissue obtained at surgery, consisting of 18 cases of papillary carcinoma, 9 cases of follicular carcinoma, and 9 cases of anaplastic carcinoma were fixed in formalin solution, and paraffin sections were made. They were stained for PECAM-1 using the avidin-biotin complex (ABC) technique. The volume of the blood vessels and their three-dimensional (3D) structure were analyzed using an image analyzer. The volume ratios of blood vessels in thyroid tissues were: normal tissues, 1.10%; papillary carcinoma, 3.01%; follicular carcinoma, 8.13%; and anaplastic carcinoma, 0.91%. Ratios in malignant tumors were larger than in normal tissues, except for anaplastic carcinoma. The typical 3D structure of micro-blood vessels was histopathologically varied: branching tree-like blood vessels in papillary carcinomas; vessels of varied diameter surrounding follicle structure in follicular carcinomas; and simple and immature vessels in anaplastic carcinomas. The volume and 3D structure of micro-blood vessels in thyroid malignant tumors differed from those in normal tissues, and varied according to histological classification.     

Enhanced expression of nicotinamide N-methyltransferase in human papillary thyroid carcinoma cells

To gain an understanding of the molecular pathogenesis of thyroid cancer, we used DNA microarray to study the expression profiles of 10 different human thyroid carcinoma cell lines. These included papillary lines BHP 2-7, BHP 7-13, BHP 10-3, BHP 18-21, NPA 87, and TPC1; anaplastic lines ARO 81-1 and DRO 90-1; follicular line WRO 82-1; and medullary line HRO 85-1. Among the genes with increased expression in the cancer cell lines, a gene coding for nicotinamide N-methyltransferase (NNMT) was identified for being highly expressed only in the papillary cell lines. NNMT catalyzes N-methylation of nicotinamide and other structurally related compounds and is highly expressed in the human liver. The results were further confirmed by semiquantitative RT-PCR and Northern blot analysis. NNMT catalytic activities were determined in all of the cells described above and in additional cell lines. Significantly higher NNMT enzyme activities were detected in eight of 10 of the papillary lines and three of six of the follicular cell lines tested. Normal thyroid tissue, thyroid primary cultures, anaplastic cancer cells, and medullary cancer cells showed no or low enzyme activity. Immunohistochemical staining for NNMT of human thyroid specimens showed strong and abundant cytoplasmic reactions in the sections of papillary carcinomas, and weak or scanty reaction in the normal thyroid tissues. These results indicate that NNMT is a potential biomarker for papillary thyroid carcinoma.     

BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas

Activating point mutations of the BRAF gene have been recently reported in papillary thyroid carcinomas. In this study, we analyzed 320 thyroid tumors and six anaplastic carcinoma cell lines and detected BRAF mutations in 45 (38%) papillary carcinomas, two (13%) poorly-differentiated carcinomas, three (10%) anaplastic carcinomas, and five (83%) thyroid anaplastic carcinoma cell lines but not in follicular, Hürthle cell, and medullary carcinomas, follicular and Hürthle cell adenomas, or benign hyperplastic nodules. All mutations involved a T-->A transversion at nucleotide 1796. In papillary carcinomas, BRAF mutations were associated with older age, classic papillary carcinoma or tall cell variant histology, extrathyroidal extension, and more frequent presentation at stages III and IV. All BRAF-positive poorly differentiated and anaplastic carcinomas contained areas of preexisting papillary carcinoma, and mutation was present in both the well-differentiated and dedifferentiated components. These data indicate that BRAF mutations are restricted to papillary carcinomas and poorly differentiated and anaplastic carcinomas arising from papillary carcinomas. They are associated with distinct phenotypical and biological properties of papillary carcinomas and may participate in progression to poorly differentiated and anaplastic carcinomas.     

Identifying differentially expressed genes associated with metastasis of follicular thyroid cancer by cDNA expression array.

Patients with follicular thyroid carcinoma have a higher incidence of metastasis than papillary thyroid carcinoma when thyroid cancer is diagnosed. The cDNA expression array technology is utilized herein to profile differentially expressed genes from metastatic human follicular thyroid carcinoma and reveal new tumor markers as well as target genes for therapeutic intervention. Tissue samples were obtained during surgical resection of the thyroid follicular carcinoma and metastatic tissue in the brain of the same patient. Two identical Atlas human cDNA expression arrays were hybridized with 32P-labeled cDNA probes derived from RNA of either primary thyroid cancer or metastatic tissue. Parallel analysis of the hybridized signals allowed us to identify the alteration of gene expression in the metastasis process. Eighteen genes significantly overexpressed and 40 genes significantly underexpressed were identified in the metastatic thyroid cancer. Genes that displayed an altered expression were associated with the processes of cell cycle regulation, apoptosis, DNA damage response, angiogenesis, cell adhesion and mobility, invasion, and immune response. An expression profile of genes that are associated with metastasis process of follicular thyroid cancer was also discussed. Further investigation is required to understand the precise relationship between the altered expression of these genes and the metastasis process of follicular thyroid cancer.     

Transforming events in thyroid tumorigenesis and their association with follicular lesions

Thyroid tumors comprise a broad spectrum of neoplastic phenotypes, and distinct molecular events have been implicated in their pathogenesis. Pituitary tumor transforming gene, originally isolated from GH(4) pituitary cells, is tumorigenic in vivo, regulates basic fibroblast growth factor secretion, and is homologous to a securin inhibitor of chromatid separation. Pituitary tumor transforming gene 1 is expressed at low levels in several normal human tissues and is abundantly expressed in neoplasms, including colorectal carcinoma, where pituitary tumor transforming gene expression correlated highly with tumor invasiveness. As pituitary tumor transforming gene is regulated by E and as thyroid cancer shows a strong female preponderance, we examined pituitary tumor transforming gene 1 expression and action in human thyroid tumors and in normal human and rat thyroid cells. Increased pituitary tumor transforming gene 1 expression was evident early in thyroid tumors and was most abundantly expressed in a subset of thyroid hyperplasia, follicular adenomas, and follicular carcinomas (1.8-fold; P < 0.0001). Pituitary tumor transforming gene 1 overexpression in rat FRTL5 thyroid cells and in primary human thyroid cell cultures causes in vitro transformation and produces a dedifferentiated neoplastic phenotype. As pituitary tumor transforming gene 1 was abundantly overexpressed in follicular adenoma and follicular carcinoma, we propose that pituitary tumor transforming gene overexpression may play a role in the early molecular events leading to divergent development of follicular and papillary carcinoma.     

Epigenetic dysregulation in thyroid neoplasia.

Gain-of-function mutations in oncogenes have aided our understanding of the molecular mechanisms of thyroid carcinogenesis. Mutations or deletions cause inactivation of tumor suppressor genes in thyroid carcinomas. However, recent advances have disclosed the significance of epigenetic events in the development and progression of human tumorigenesis. Indeed, various tumor-suppressor genes and thyroid hormone-related genes are epigenetically silenced in thyroid tumors. This article reviews the evidence for epigenetic gene dysregulation in follicular cell-derived thyroid carcinomas including papillary thyroid carcinoma, follicular thyroid carcinoma, and undifferentiated thyroid carcinoma. The authors also discuss future applications of epigenetics as ancillary diagnostic tools and in the design of targeted therapies for thyroid cancer.     

Maspin expression is directly associated with biological aggressiveness of thyroid carcinoma.

Maspin belongs to the serpin superfamily and has been identified as a tumor suppressor because it inhibits cell motility, invasion, and angiogenesis. However, its physiological activity in carcinoma tissues seems to differ according to the origin of the carcinoma. In this study, we investigated maspin expression in thyroid neoplasms originating in follicular cells by means of immunohistochemistry. Neither normal follicular cells nor stromal cells expressed maspin. Follicular adenomas were all negative for maspin, but 12.5% of follicular carcinomas and 30.5% of papillary carcinomas were positive for it. However, the staining pattern was only focal in all positive specimens except for one and maspin-expressing cells were present mainly at the edge of carcinoma nests. Widely invasive follicular carcinomas tended to be more frequently positive for maspin than minimally invasive ones. In papillary carcinoma, maspin expression was directly linked to stage, tumor size, and extrathyroidal invasion. Papillary and follicular carcinomas involving lesions with solid, trabecular, or scirrhous growth patterns (poorly differentiated carcinoma as designated by Sakamoto et al.) expressed maspin in significantly higher incidence than those with pure papillary or follicular patterns. Furthermore, 48.2% of anaplastic (undifferentiated) carcinomas diffusely expressed maspin, and the rest were completely negative. These findings indicate that, in contrast to other carcinomas, maspin expression is directly associated with the biological aggressiveness of thyroid carcinoma. Further studies regarding the function of maspin in this carcinoma are required.     

Clinical implication of hot spot BRAF mutation,V599E,in papillary thyroid cancers

Activating mutations in the BRAF kinase gene have recently been reported in human cancers. The aim of the present study was to determine the frequency of BRAF mutations in thyroid cancer and their correlation with clinicopathological parameters. We analyzed exons 11 and 15 of BRAF gene in six human thyroid cancer cell lines and 207 paraffin-embedded thyroid tumor tissues. A missense mutation was found at T1796A (V599E) in exon 15 in four of the six cell lines and 51 of 207 thyroid tumors (24.6%; 0 of 20 follicular adenoma, 0 of 11 follicular carcinoma, 49 of 170 papillary carcinomas, and 2 of 6 undifferentiated carcinomas). Activation of MAPK kinase-MAPK pathway was observed in cell lines harboring BRAF mutation. BRAF mutation-associated enhanced cell growth was suppressed by MAPK kinase inhibitor, U0126. Examination of 126 patients with papillary thyroid cancer showed that BRAF mutation correlated significantly with distant metastasis (P = 0.033) and clinical stage (P = 0.049). Our results indicate that activating mutation of BRAF gene could be a potentially useful marker of prognosis of patients with advanced thyroid cancers.     

The antiapoptotic protein BAG3 is expressed in thyroid carcinomas and modulates apoptosis mediated by tumor necrosis factor-related apoptosis-inducing ligand

CONTEXT: We previously showed that BAG3 protein, a member of the BAG (Bcl-2-associated athanogene) co-chaperone family, modulates apoptosis in human leukemias. The expression of BAG3 in other tumor types has not been extensively investigated so far. OBJECTIVE: The objective of this study was to analyze BAG3 expression in thyroid neoplastic cells and investigate its influence in cell apoptotic response to TNF-related apoptosis-inducing ligand (TRAIL). DESIGN, SETTING, AND PATIENTS: We investigated BAG3 expression in human thyroid carcinoma cell lines, including NPA, and the effect of BAG3-specific small interfering RNA on TRAIL-induced apoptosis in NPA cells. Subsequently, we analyzed BAG3 expression in 30 benign lesions and 56 carcinomas from patients of the Naples Tumor Institute Fondazione Senatore Pascale. MAIN OUTCOME MEASURES: The main outcome measures were: analysis of BAG3 protein in NPA cells by Western blot and immunocytochemistry; analysis of apoptosis in TRAIL-stimulated NPA cells by flow cytometry; and evaluation of BAG3 expression in specimens from thyroid lesions by immunohistochemistry. RESULTS: BAG3 was expressed in human thyroid carcinoma cell lines; small interfering RNA-mediated downmodulation of its levels significantly (P < 0.0195) enhanced NPA cell apoptotic response to TRAIL. The protein was not detectable in 19 of 20 specimens of normal thyroid or goiters, whereas 54 of 56 analyzed carcinomas (15 follicular, 28 papillary, and 13 anaplastic) were clearly positive for BAG3 expression. CONCLUSIONS: BAG3 downmodulates the apoptotic response to TRAIL in human neoplastic thyroid cells. The protein is specifically expressed in thyroid carcinomas and not in normal thyroid tissue or goiter.     

Expression of tpo mRNA in thyroid tumors: quantitative PCR analysis and correlation with alterations of ret, Braf , ras and pax8 genes

Immunocytochemistry (ICC) of thyroid peroxidase (TPO) using the monoclonal antibody MoAb47 has been used as malignancy marker on thyroid fine needle aspiration. However, little is known about the fate of TPO in thyroid carcinoma. We performed a qualitative PCR (Q-PCR) analysis to measure the expression of variants of tpo mRNA in 13 normal tissue samples, 30 benign tumors (BT), 21 follicular carcinomas (FC), 20 classical papillary carcinomas (PCc), 12 follicular variants of papillary carcinomas (PCfv) and nine oncocytic carcinomas (OC). We also studied mutations involving the ras, Braf, ret or pax8 genes. Results of Q-PCR were closely correlated with those of ICC (P < 0.0001; R = 0.59) and showed that overall tpo expression was lower in all carcinomas than in normal and BT (P < 0.05). The ratio tpo2 or tpo3 to tpo1 was inversed in follicular tumors. Genetic mutations were observed in 90% of PCc, 61.9% of FC, 41.7% of PCfv, 0% of OC and 10% in BT. pax8-ppar gamma1 rearrangement was correlated with qualitative changes in tpo mRNA (P < 0.01). These results confirmed the decrease of TPO expression in 97% of thyroid carcinomas regardless of histological type and the overexpression of shorter splice variants in follicular tumors. Both reduction in quantity of TPO and impairment of its maturation process could account for the atypical immunohistochemical reaction of MoAb47 with TPO.     

Expression and localization of the homeodomain-containing protein HEX in human thyroid tumors

Homeobox genes are involved in neoplastic transformation of both epithelial and hemopoietic tissues. The divergent homeobox gene HEX is expressed in the anterior visceral endoderm during early mouse development and in some adult tissues of endodermal origin, including liver and thyroid. Whereas a role in leukemyogenesis has been proposed already, few data are available on the involvement of HEX in human epithelial tumors. Herein, we analyzed HEX expression and subcellular localization in a series of 55 human thyroid tumors and in several tumoral cell lines. HEX mRNA was detected by RT-PCR either in normal tissues or in thyroid adenomas and differentiated (papillary and follicular) carcinomas. HEX mRNA was also expressed in most undifferentiated carcinomas. Subcellular localization of HEX protein was investigated by immunohistochemistry. In normal tissues and adenomas, HEX protein was present both in nucleus and cytoplasm. In contrast, both differentiated and undifferentiated carcinomas, as well as the tumoral cell lines investigated, showed HEX protein only in the cytoplasm. These findings suggest that regulation of HEX entry in the nucleus of thyrocytes may represent a critical step during human thyroid tumorigenesis.     

Molecular profiling distinguishes papillary carcinoma from benign thyroid nodules

Recently we identified a molecular basis for differentiating benign and malignant follicular thyroid tumors. The purpose of these studies was to determine whether molecular analysis can be used to differentiate papillary thyroid carcinomas from benign thyroid nodules. Gene expression patterns of 14 papillary thyroid carcinomas and 21 benign tumors were analyzed by oligonucleotide array analysis. The carcinomas included seven classical papillary thyroid carcinomas (PTC) and seven follicular variant of PTC (FVPTC), and the benign tumors included 14 follicular adenomas and seven hyperplastic nodules. A hierarchical clustering analysis was performed to examine the groups for potential differences. The combined PTC and FVPTC groups had a distinct gene expression profile compared with the benign lesions. The sensitivity for a diagnosis of carcinoma was 93%, with a 100% specificity (one FVPTC clustered with the benign nodules). Cancer gene profiles contained both known (Met and galectin-3) and previously unidentified genes. Gene profiling is a reliable means of distinguishing PTC, FVPTC, and benign tumors of the thyroid. These gene profiles may provide insight into the pathogenesis of papillary thyroid carcinoma and may ultimately enhance the preoperative diagnosis of thyroid nodules on a molecular basis.