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Dipartimento di Medicina Sperimentale (E.F., A.P., M.S.E., I.S., A.G.) and Scienze Cliniche (E.T., A.S., R.M., C.D., S.F.) and Istituto Pasteur (I.S., A.G.), Fondazione Cenci-Bolognetti, University of Rome La Sapienza, 00161 Rome, Italy; Neuromed Institute (A.G.), 86077 Pozzilli (Isernia), Italy; Dipartimento di Scienze Farmacobiologiche (D.R.), University of Catanzaro Magna Græcia, 88100 Catanzaro, Italy; and Department of Nuclear Medicine and Endocrine Oncology (M.S.), Institut Gustave Roussy and University Paris-Sud 11, 94805 Villejuif Cedex, France
Address all correspondence and requests for reprints to: Sebastiano Filetti, M.D., 2a Clinica Medica, Dipartimento di Scienze Cliniche, Università degli Studi di Roma La Sapienza, V.le del Policlinico, 155, 00161 Rome, Italy. E-mail: sebastiano.filetti{at}uniroma1.it; or Alberto Gulino, Dipartimento di Medicina Sperimentale, Istituto Pasteur, Fondazione Cenci-Bolognetti, Università degli Studi di Roma La Sapienza, V.le del Policlinico, 155, 00161 Rome, Italy. E-mail: alberto.gulino{at}uniroma1.it.
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Abstract |
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Objective: The objective of the study was to characterize the expression of Notch pathway components in thyroid follicular cells and Notch signaling activities in normal and transformed thyrocytes.
Design/Setting and Patients: Expression of Notch pathway components and key markers of thyrocyte differentiation was analyzed in murine and human thyroid tissues (normal and tumoral) by quantitative RT-PCR and immunohistochemistry. The effects of Notch overexpression in human thyroid cancer cells and FTRL-5 cells were explored with analysis of gene expression, proliferation assays, and experiments involving transfection of a luciferase reporter construct containing human NIS promoter regions.
Results: Notch receptors are expressed during the development of murine thyrocytes, and their expression levels parallel those of thyroid differentiation markers. Notch signaling characterized also normal adult thyrocytes and is regulated by TSH. Notch pathway components are variably expressed in human normal thyroid tissue and thyroid tumors, but expression levels are clearly reduced in undifferentiated tumors. Overexpression of Notch-1 in thyroid cancer cells restores differentiation, reduces cell growth rates, and stimulates NIS expression via a direct action on the NIS promoter.
Conclusion: Notch signaling is involved in the determination of thyroid cell fate and is a direct regulator of thyroid-specific gene expression. Its deregulation may contribute to the loss of differentiation associated with thyroid tumorigenesis.
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Introduction |
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-like 1, 3, 4, and Jagged-1 and -2). Once activated, Notch receptors are cleaved by the 
-secretase protease complex. This releases a cytoplasmic domain fragment, which translocates into the nucleus and modulates gene transcription, mainly after binding to C-promoter binding factor transcription factors (2). Several Notch target genes have been identified (1, 3), including basic helix-loop-helix transcription factors belonging to the Hes1 (enhancer of split-homolog-1) and Herp and Hrt/Hey (Hes-related repressor protein) families. A number of other proteins modulate Notch signaling, and Notch receptors can also directly activate signal transduction pathways in the cytoplasm using the E3 ubiquitin ligase Deltex protein (1). The Notch pathway plays fundamental roles in embryonic development and the regulation of self-renewing tissues (4). It is also known to exert oncogenic or tumor suppressor effects, depending on the level of signaling activity and the context in which it occurs (cell type, presence or absence of the specific growth factors in the microenvironment) (5). The role of Notch signaling in controlling the growth and differentiation of thyroid follicular cells has not been fully published. Information on thyroid development in the absence of Notch1 or mammalian Hes1 expression is not available because disruption of these genes is associated with embryonic lethality (6, 7), and the effects of increased Notch activity in thyrocytes have not been elucidated.
In this study, we analyzed the expression of components of the Notch pathway in thyroid follicular cells and evaluated the direct functional consequences of Notch signaling in normal and transformed thyrocytes. We found that components of this pathway are expressed in normal thyroid cells and that their expression is up-regulated by TSH and decreased in anaplastic thyroid tumors. In addition, cell transfection experiments showed that overexpression of Notch1 or Hes1 in thyroid cancer cells reduced cell growth and restored expression levels of most thyroid differentiation markers. Overexpression of Notch pathway components was also found to cause direct activation of the promoter region of the sodium-iodide symporter (NIS) gene.
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Materials and Methods |
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Normal thyroid glands were obtained from 6-, 12-, and 60-d-old CD1 mice (Charles River, Calco, Lecco, Italy).
Human thyroid tissue samples
Samples of human thyroid tumors were collected from 28 patients undergoing surgery for thyroid cancer at the University of Rome Medical Center or the Institut Gustave Roussy. The tissues were snap frozen and stored at –80 C until use. The tumors were classified according to World Health Organization histological criteria (8) as thyroid papillary carcinoma (PTC) (n = 12), follicular carcinoma (FTC) (n = 8), or anaplastic carcinoma (ATC) (n = 8). For each tumor sampled, normal thyroid was collected from the same patient. We also examined additional 20 samples randomly selected from a series of normal thyroid glands collected during a previous study (9). These tissues came from patients operated on for benign thyroid nodules, whose serum TSH levels at the time of surgery were within normal limits (1.0–4.0 mU/liter) or less than 0.5 mU/liter as a result of suppressive L-T4 therapy. Sample aliquots were either immersed in liquid nitrogen and stored at –70 C or fixed in 10% formalin until analysis.
The study protocol was approved by the local medical ethics committee. Before surgery, each study participant provided written, informed consent to the collection of fresh thyroid tissue for genetic studies.
Cell cultures
We used human ARO and WRO cells (American Type Culture Collection, Manassas, VA) as models of ATC and FTC, respectively. The cells were cultured in DMEM containing 10% fetal bovine serum, as previously described (10). Controls consisted in normal rat thyroid cells of the FRTL-5 line cultured in Coon’s modified F12 medium with 5% calf serum and a mixture of six hormones (11).
Cell transfection, luciferase assays, and Western blot
Cells were transfected with 1 µg of the Notch1 intracellular domain (N1-ICD) or the Hes1 gene subcloned in-frame into the mammalian expression vector pcDNA3.1 (Invitrogen, San Giuliano Milanese, Milan, Italy). The TransFast transfection reagent (Promega, Milan, Italy) was used in accordance with the manufacturer’s instructions. In some experiments, cells were cotransfected with 0.05 µg of luciferase reporter and Renilla-expressing vector pRL-TK, (Promega, Madison, WI) or an NIS-LUC vector (kindly provided by Professor R. Di Lauro, Stazione Zoologica Anton Dohrn and Department of Cellular and Molecular Biology and Pathology, University of Naples "Federico II", 80727 Naples, Italy) (12) containing the Hes1-binding sites of the promoter region of the rat NIS gene (rNIS promoter_LUC). Reporter activity was measured with a dual luciferase assay system (Promega) 24 h after transfection with 0.5 µg of total plasmid DNA per well, and the results are presented as means ± SD of values from at least three experiments, each performed in triplicate.
Transfected cells were lysed in a solution containing Tris-HCl (pH 7.6) 50 mM, deoxycholic acid sodium salt 1%, NaCl 150 mM, Nonidet P-40 1%, EDTA 5 mM, NaF 100 mM, and protease inhibitors. Lysates were centrifuged at 13,000 x g for 20 min and separated on SDS-PAGE and immunoblotted using standard procedures. Anti-Notch1 C20 antibody (sc-6014; Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:500 and a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) diluted 1:5000 were used, followed by enhanced chemiluminescence detection system (ECL Plus; Amersham-GE Healthcare, Piscataway, NJ).
Cell growth assays
Cell proliferation was evaluated 24 h after transfection by means of cell counts, trypan blue exclusion assay, and bromodeoxyuridine (BrdU) labeling assays (Roche, Mannheim, Germany), as previously described (13).
Immunohistochemistry
Sections (5 µm thick) cut from paraffin-embedded tissue blocks were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked by treatment with 0.03% H2O2 Tris-HCL buffer for 10 min, and the sections were microwaved three times (5 min each time) in EDTA buffer to retrieve antigens. The tissues were blocked with 2% goat serum (Sigma Aldrich, Milan, Italy) and immunostained for 90 min at room temperature with a polyclonal antibody against Notch1 (C20 sc-6014; SantaCruz Biotechnology) diluted 1:100 or with a primary polyclonal antibody against the NIS (14) diluted 1:1000. After washes with Tris-HCL 1x buffer, secondary biotinylated antibodies (Vector Laboratories, Milan, Italy) were applied. Binding of antibodies was evaluated after application of the biotin-avidin-based Vectastain Elite ABC reagent (Vector Laboratories). Diaminobenzidine (DakoCytomation, Milan, Italy) was used as the final chromogen, and hematoxylin was used to counterstain nuclei. Negative controls for all antibodies were made by replacing the primary antibody with nonimmunogenic IgG.
Real-time RT-PCR
Total RNA was isolated with Trizol reagent (Invitrogen), and 1 µg of each sample was reverse transcribed with Superscript II reverse transcriptase and random hexamers (Invitrogen). The cDNA samples were diluted 10-fold, and real time PCRs were carried out for each gene being studied with Assay on Demand Taqman reagents (Applied Biosystems, Foster City, CA). Amplifications were performed in an ABI Prism 7900 sequence detection system (Applied Biosystems). A reaction mixture containing cDNA template, TaqMan universal PCR master mix (Applied Biosystems), and the primer probe mixture was amplified using standard quantitative PCR thermal cycler parameters. Each amplification reaction was performed in triplicate, and the average of the three threshold cycles was used to calculate the amount of transcript in the sample (using SDS version 1.7a software; Applied Biosystems, Foster City, CA). mRNA quantification was expressed in arbitrary units as the ratio of the sample quantity to the quantity of the calibrator. All values were normalized with three endogenous controls, glyceraldehyde-3-phosphate dehydrogenase, β-actin, and hypoxanthine-guanine phosphoribosyl transferase, which yielded similar results.
Statistical analysis
Results are expressed as mean ± SD from an appropriate number of experiments, as indicated in the figure legends. Statistical analysis was based on the Mann-Whitney U test for nonparametric values and was carried out with StatView 4.1 software (Abacus Concepts, Berkeley, CA). P < 0.05 was considered significant.
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Results |
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Immunohistochemical analysis of Notch1 expression in murine thyroid tissues at different stages of postnatal development revealed positive staining on postnatal d 6. Expression increased thereafter, and nuclear staining was clearly observed through postnatal d 60 (Fig. 1
). Interestingly, the increasing expression of nuclear Notch1 was paralleled by that of the NIS protein, located in the cell plasma membrane (Fig. 1).
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). Similar patterns were observed in normal human thyroid tissues from adults with normal or below-normal serum levels of TSH. Transcripts for Notch1, 2, 3, and Hes1 were present in both sets of tissues, but the tissues from patients subjected to TSH suppression were characterized by lower mRNA for all Notch proteins (except Notch2) and Hes1 (Fig. 2B). Again, expression of Notch signaling components paralleled that of the NIS gene (Fig. 2B) and genes for other thyroid-specific markers, such as thyroperoxidase, TSH receptor, apical iodide transporter B, and thyroglobulin.
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Next, we investigated the expression of the Notch pathway components in thyroid tumor tissues and, for some cases, the matched normal controls. Quantitative real-time RT-PCR assays revealed histotype-related variability in the expression of the three Notch genes in the different tumor histotypes. However, compared with PTCs (n = 12) and FTCs (n = 8), ATCs (n = 8) exhibited significantly decreased expression of mRNA for Notch1 and -3 and Hes1 (Fig. 3A). Because Hes1 is one of the main effectors of the Notch pathway, the latter change seems to reflect the deregulation of this pathway in thyroid tumors. NIS mRNA levels were clearly decreased in all three tumor types, although the most marked reductions were seen in ATCs (Fig. 3A). In immunohistochemical studies, all three tumor histotypes also displayed decreased Notch1 protein expression, compared with normal thyroid tissues, which was consistent with the down-regulated expression of the Notch signaling genes and their effector Hes1 (Fig. 3B).
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ARO cells, which normally express only low levels of Notch1 and Hes1 (data not shown), were transiently transfected with the N1-ICD cDNA that had been cloned into a mammalian expression vector. These transfected cells displayed increased expression of Hes1 mRNA and near-normal expression of NIS and thyroperoxidase (TPO), but not thyroglobulin (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org). We suspected that the latter effects might be mediated by increased expression of thyroid transcription factor-1 and paired box transcription factor-8, transcription factors known to exert direct actions on the NIS and TPO gene promoters. However, the transcripts levels of these two factors in transfected cells were not significantly different from those observed in normal ARO cells (supplemental Fig. 1). Similar results were obtained with FRTL-5 cells, in which overexpression of the Notch1 gene increased the expression of NIS, TPO, and Hes1 (supplemental Fig. 1).
Notch1 and Hes1 activate NIS transcription
The findings outlined above suggested that activation of Notch signaling might exert a direct effect on NIS promoter activity. To test this hypothesis, we analyzed the NIS promoter region for the presence of putative Hes1-responsive sites (N-box). These studies revealed two potential Hes1 binding sites in the human promoter (supplemental Fig. 2) and six in the promoter region of the rat analog (data not shown). Next, we cotransfected cells with Hes1 or Notch1 vectors plus a luciferase reporter construct with the regions of the rat NIS promoter that contained the Hes1-binding sites (rNIS promoter-LUC) (12). Transfection of N1-ICD plasmid resulted in induction of Hes1 (mRNA), as shown in Fig. 4A. In luciferase assays, cotransfected ARO, WRO, and FRTL-5 cells all exhibited increases in the NIS-promoter activity (Fig. 4B). These results indicate that the NIS gene is a direct target of Notch signaling and is transcriptionally activated by Hes1.
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FRTL-5 cells transfected with Notch1 were then treated with a -secretase inhibitor (Z-LLNIe-CHO; Calbiochem, Merck Chemicals Ltd, Darmstadt, Germany) for 48 h to abolish Notch signaling. As shown in Fig. 5A, mRNA levels for Hes1 and NIS were significantly decreased after 24 and 48 h of treatment with this drug (1 and 3 µM). The effect was reversible, and transcript levels for both genes increased progressively during the 120 h after suspension of the treatment (Fig. 5B).
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Finally, we investigated the effects of Notch pathway activation on the growth of ARO cells, compared with that of normal thyroid cells (FRTL-5 cell line). Cells transfected with Notch1 exhibited significantly lower proliferation rates than those observed when transfection was done with vector alone (P < 0.01), in both tumoral (Fig. 5
, A and B) and normal (Fig. 5, C and D) thyroid cells. Notch1 overexpression has been controlled by Western blot (supplemental Fig. 3). The results that emerged when proliferation was evaluated with the trypan blue exclusion assay were similar to those measured by means of BrdU incorporation (Fig. 6). With the former assay, an increase of cell mortality also was detectable in transfected cells vs. control cells (
1.5-fold over control after 24 h, 5-fold after 48 h) (data not shown).
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Discussion |
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Various attempts for combating cancer are being made to find novel molecules that are capable of not only blocking tumor cell growth but also restoring a differentiated phenotype. For thyroid cancer cells, this would mean restoring (among other things) the iodine-concentrating capacity that renders such cells amenable to radioiodine treatment, and in this context, great interest has been aroused by attempts to enhance NIS expression in transformed thyrocytes (15, 16). Our data demonstrate that the elements of Notch cascade are variably expressed in thyroid tumor tissues, but the activity of this pathway is clearly down-regulated in undifferentiated thyroid tumors. Even more interesting is our finding that overexpression of Notch1 in thyroid cancer cells restores normal expression levels of several markers of thyrocyte differentiation and also reduces the growth rate of these cells. Our demonstration that the Notch pathway effector Hes1 exerts a direct action on the promoter region of the NIS gene is particularly intriguing because a better understanding of the transcriptional regulation of this gene is a fundamental prerequisite for attempts to restore the ability of thyroid tumor cells to concentrate radioiodine.
Whether Notch signaling produces oncogenic or tumor suppressor effects seems to depend largely on the cell type in which it occurs. It has been hypothesized that up-regulated Notch expression promotes tumorigenesis in cell contexts in which it normally functions as a stem cell gatekeeper or as a determinant of precursor cell fate, whereas it is likely to have tumor suppressor effects in tissues in which it initiates terminal differentiation events (17). Aberrant expression of Notch receptors, ligands, and targets is observed in many hematopoietic and solid tumors, and it is often associated with poor prognosis or metastatic disease (1, 3). Inhibition of Notch signaling is therefore emerging as a very promising antitumoral strategy, in large part because of its potential effects on cancer stem cells, which are known to express high levels of Notch1 (18). Our findings, however, indicate that Notch signaling in thyroid follicular cells tends to suppress tumorigenesis, an effect first described in keratinocytes (19) and subsequently documented in other cell types, including prostate cancer, small cell lung cancer, pancreatic carcinoid, hepatocellular carcinoma, cervical cancer cells, B cells, and other hematopoietic lineages in vitro (20, 21, 22, 23, 24, 25, 26). Activated Notch1 has also been shown to inhibit proliferation and alter the neuroendocrine phenotype of medullary thyroid cancer cells (27). It is important to consider, however, that most of these studies used transient expression with very high levels of active Notch1. Some investigators have hypothesized that Notch1, like p53, activates different targets, depending on whether it is expressed at high or low levels, and that it is thus capable of stimulating or arresting cell growth in different situations (3). These findings underline the need for caution and close monitoring in the assessment of Notch inhibitors as novel anticancer agents (28). The potential toxicity of these agents is largely unexplored, and they may have adverse effects in cells in which normal Notch signaling inhibits growth and promotes differentiation, including keratinocytes, parafollicular thyroid cells, and, in light of the results of the present study, thyrocytes as well.
While the present study was being completed, Mitsutake et al. (29) described increased Jag1 and Hes1 gene expression in side-population cells (a subtype of cells showing the characteristic of normal stem cells) obtained from human thyroid cancer cell cultures. This finding appears to be in conflict with our observation of reduced Notch-pathway activity in less differentiated thyroid tumors. However, it is important to note that the side-population subset described by Mitsutake et al. accounted for less than 1% of the total cells in their cultures, and this might explain the apparent discordance with our data.
In conclusion, this report provides the first description of the expression of Notch pathway components in thyroid follicular cells and the role played by Notch signaling in thyrocyte differentiation. The Notch cascade may represent a novel target for attempts to achieve redifferentiation (partial or complete) of thyroid tumor cells. The fact that Notch signaling seems capable of inducing the expression of the NIS gene is particularly intriguing because restoration of NIS activity could render less differentiated thyroid carcinomas amenable to radioiodine therapy.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online July 29, 2008
Abbreviations: ATC, Anaplastic carcinoma; BrdU, bromodeoxyuridine; FTC, follicular carcinoma; N1-ICD, Notch1 intracellular domain; NIS, sodium-iodide symporter; PTC, papillary carcinoma; TPO, thyroperoxidase.
Received March 5, 2008.
Accepted July 23, 2008.
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