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. 2013 Apr 1;6(2):197–205. doi: 10.1593/tlo.12442

Notch Pathway Is Activated by MAPK Signaling and Influences Papillary Thyroid Cancer Proliferation1,2

Alex Shimura Yamashita 1, Murilo Vieira Geraldo 1, Cesar Seigi Fuziwara 1, Marco Aurélio Vamondes Kulcsar 1, Celso Ubirajara Moretto Friguglietti 1, Ricardo Borges da Costa 1, Gilson Soares Baia 1, Edna Teruko Kimura 1
PMCID: PMC3610552  PMID: 23544172

Abstract

Mutually exclusive genetic alterations in the RET, RAS, or BRAF genes, which result in constitutively active mitogen-activated protein kinase (MAPK) signaling, are present in about 70% of papillary thyroid carcinomas (PTCs). However, the effect of MAPK activation on other signaling pathways involved in oncogenic transformation, such as Notch, remains unclear. In this study, we tested the hypothesis that the MAPK pathway regulates Notch signaling and that Notch signaling plays a role in PTC cell proliferation. Conditional induction of MAPK signaling oncogenes RET/PTC3 or BRAFT1799A in normal rat thyroid cell line mediated activation of Notch signaling, upregulating Notch1 receptor and Hes1, the downstream effector of Notch pathway. Conversely, pharmacological inhibition of MAPK reduced Notch signaling in PTC cell. Thyroid tumor samples from transgenic mice expressing BRAFT1799A and primary human PTC samples showed high levels of Notch1 expression. Down-regulation of Notch signaling by γ-secretase inhibitor (GSI) or NOTCH1 RNA interference reduces PTC cell proliferation. Moreover, the combination of GSI with a MAPK inhibitor enhanced the growth suppression in PTC cells. This study revealed that RET/PTC and BRAFT1799A activate Notch signaling and promote tumor growth in thyroid follicular cell. Taken together, these data suggest that Notch signaling may be explored as an adjuvant therapy for thyroid papillary cancer.

Introduction

Thyroid cancer is the most common endocrine malignancy and its incidence continues to increase [1]. Papillary thyroid carcinoma (PTC) is the most prevalent type of thyroid cancer accounting for 80% of cases [1–3]. In PTC, genetic alterations in mitogen-activated protein kinase (MAPK) signaling components such as RET/PTC, RAS, and BRAF are well studied and result in constitutive activation of the MAPK signaling pathway [4–7]. The RET/PTC and BRAFT1799A oncogenes are involved in thyroid tumorigenesis as demonstrated by targeted expression of RET/PTC or BRAFT1799A oncogenes in transgenic mice, suggesting that mutations in MAPK signaling components contribute for transformation to PTC [8,9]. However, the mechanism of concomitant activation of different signaling pathways by these oncogenes in thyroid cancer is not fully understood.

Notch signaling is critical for the development and maintenance of tissue homeostasis [10]. The Notch signaling pathway comprises a family of transmembrane receptors and their ligands; to date, four mammalian receptors (Notch1, 2, 3, and 4) and at least five ligands [Delta 1, 3, and 4 and Jagged (Jag) 1 and 2] have been identified. Binding of the ligand renders the Notch receptor susceptible to sequential proteolytic cleavage mediated by ADAM metalloprotease and γ-secretase enzymes, which in turn results in the release of the Notch intracellular domain from the plasma membrane and its subsequent translocation into the nucleus [10,11]. Notch intracellular domains function within the nucleus as co-activators with the CBF1/RBPjκ in mammalian, Suppressor of Hairless Su(H) in Drosophila melanogaster, Lag1 in Caenorhabditis elegans family of transcription factors to promote transcription of target genes such as hairy and enhancer of split 1 (HES1) and hair/enhancer of split related with YRPW motif [12]. Aberrant Notch signaling has been linked to a wide variety of tumor types and can either suppress or promote tumorigenesis depending on the cell type and context. Activated Notch has been shown to transform primary Schwann cells [13], melanocytes [14], and epithelial breast cells [15]. Notch signaling dysregulation has been observed in small cell lung cancer, neuroblastoma, and breast, cervical, and prostate carcinoma [16–20]. In PTC, a large-scale gene expression analysis showed enhanced gene expression of several components of Notch signaling [21].

Growing evidence indicates that MAPK signaling pathway impacts Notch signaling. For instance, the expression of mutated RasV12 up-regulates Notch1 protein expression in fibroblast and epithelial human cell lines, which suggests Notch as a key downstream target of oncogenic RAS [22]. Since activation of MAPK signaling is the most frequent oncogenic genetic alteration in PTC, we hypothesized that the two most critical oncogenes implicated in PTC tumorigenesis, RET/PTC and BRAFT1799A, regulate Notch signaling and that the Notch pathway is involved in PTC proliferation.

Materials and Methods

Cell Culture, Transfection, and Tumor Samples

The rat thyroid cell line (PCCL3) conditionally expressing either the RET/PTC3 or BRAFT1799A oncogenes, designed PTC3-5 [23] and PC-BRAF [24] cells, respectively, were maintained in Ham's F12 medium supplemented with 5% FBS, 1 mIU/ml bovine thyroid-stimulating hormone (Sigma, St Louis, MO), 10 µg/ml insulin (Sigma), 5 µg/ml apotransferrin (Sigma), 10 nM hydrocortisone (Sigma), 100 U/ml penicillin (Invitrogen Life Technologies, Carlsbad, CA), 100 µg/ml streptomycin (Invitrogen Life Technologies), and 1 µg/ml amphotericin B (Invitrogen Life Technologies). Doxycycline (Calbiochem, San Diego, CA) was used to induce RET/PTC3 and BRAFT1799A oncogenes. Human PTC cell lines (TPC-1 and BCPAP) were maintained in Dulbecco's modified Eagle's medium with 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 µg/ml amphotericin. Media for TPC-1 cells were supplemented with 5% FBS, while media for BCPAP cells were supplemented with 10% FBS. PD98059 and U0126 (Promega, Madison, WI) were used to inhibit mitogen-activated or extracellular signal-related protein kinase kinase (MEK) activity, and Z-Leu-Leu-Nle-CHO was used to inhibit-secretase activity (Calbiochem). TPC-1 cell line was transiently transfected with 10 or 30 nM of siRNA-NOTCH1 or siRNA-enhanced green fluorescent protein (EGFP) (esiRNA human NOTCH1-EHU150431; esiRNA targeting EGFP-EHUGFP; Sigma) using Lipofectamine 2000, according to the manufacturer's instructions (Invitrogen Life Technologies). Plasmid pBABE-NOTCH1 and pBABE empty vector were transfected in PCCL3 cell line to generate the PCNOTCH1 and PC-Ø, respectively, and selected with neomycin (300 µg/ml). PC-BRAF cells (1 x 104/well) were seeded into 24-well plates and co-transfected in triplicate with 300 ng of 4x CBF1-Luc and 30 ng of pRL-CMV using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. One day after transfection, cells were treated with 1 µg/ml doxycycline, and 72 hours after oncogene induction, luciferase activity was measured using the Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Human PTC and nontumor paired thyroid tissue samples were collected from patients with informed consent. This study complied with the guidelines from the ethical committee of the Institute of Biomedical Sciences (No. 193/F93/L02), University of Sao Paulo.

Cell Proliferation Assays

Human PTC cells, PC-Ø, and PC-NOTCH1 were seeded into six-well plates at a density of 1 x 104 cells/well. TPC-1 was transfected with siRNA-NOTCH1 (30 nM) or siRNA-EGFP (30 nM). Cells were enumerated using a flow cytometry-based cell counter, the Guava EasyCyte (Guava Technology, Billerica, MA). The average cell number at each time point was obtained from triplicate measurements. To assess cell viability using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, human PTC cells were seeded into 96-well plates at a density of 5 x 103 cells/well. MTT (Amresco, Solon, OH) was added to the medium at a concentration of 0.125 mg/ml and cells were solubilized in 100 µl of 0.04 M HCl in isopropanol; absorbance at 595 nm was measured using a spectrophotometer (Spectra Max Plus; Molecular Devices, Sunnyvale, CA).

Flow Cytometry

PTC cells were collected by trypsinization and double stained with fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide for 15 minutes at room temperature according to the manufacturer's instructions (Annexin V FITC Kit; Invitrogen Life Technologies) followed by analysis with a flow cytometer (Guava Technology).

Quantitative Polymerase Chain Reaction

Total RNA was extracted by phenol-chloroform method using the TRIzol reagent (Invitrogen Life Technologies). The first strand of cDNA was generated from 1 µg of total RNA in the presence of Oligo dT and MMLV reverse transcriptase (Invitrogen Life Technologies). Polymerase chain reaction (PCR) amplification was performed in duplicate using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) in an ABI 7300 Real-Time PCR System (Applied Biosystems) using primers as shown in Table W1. Gene expression was normalized to the RPL19 gene. Data were calculated using the 2-ΔΔCT method [25] and are presented as the fold change in gene expression relative to the control sample.

Western Blot Analysis

Total protein lysates were extracted using RIPA buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, and 0.1% sodium dodecyl sulfate (SDS)] containing protease inhibitor cocktail (Sigma). Protein concentration was measured using the Bradford method (Bio-Rad Laboratories, Hercules, CA) and 15 to 30 µg of each sample was separated by 10% to 12% SDS-polyacrylamide gel electrophoresis (PAGE) and blotted onto a nitrocellulose membrane (Hybond-ECL; Amersham Biosciences, Little Chalfont, United Kingdom). Nonspecific binding sites were blocked by incubating with 5% nonfat dry milk in Tris-buffered saline-0.1% Tween-20. The following primary antibodies were used: anti-ERK1 (K23), anti-phospho-extracellular signal-regulated kinase (ERK) (Tyr 204) (E4), anti-Notch1 (C20), anti-α-tubulin (B-7), anti- cyclin D1 (H-295) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and anti-α-actin (A2066) (Sigma). The antigen-antibody complexes were visualized using an HRP-conjugated secondary antibody and an enhanced chemiluminescence system (Amersham Biosciences).

Immunohistochemistry and Immunofluorescence

Immunohistochemistry to detected Notch1 protein expression was performed on formalin-fixed paraffin-embedded tissue sections by an indirect three-stage immunoenzymatic method as previously described [26]. Immunodetection of Notch1 protein (1:100, C-20; Santa Cruz Biotechnology) expression was performed on 3-µm sections and the presence of brown immunoprecipitates visible under a light microscope (Eclipse E600; Nikon, Tokyo, Japan) indicated positive immunostaining. Immunofluorescence was performed as previously described [27]. In brief, coverslip cell cultures were fixed in 3.7% paraformaldehyde, permeabilized, and blocked with 1% BSA. Cell slides were incubated with anti-Notch1 antibody (1:200, C-20; Santa Cruz Biotechnology) and anti-α-tubulin (1:500, B-7; Santa Cruz Biotechnology), then with Alexa Fluor 488-conjugated anti-rabbit and Alexa Fluor 532 anti-mouse IgG secondary antibodies (Invitrogen). The nuclei was detected with 4′,6-diamidino2-phenylindole (1 µg/ml; Sigma). Coverslip cultures were mounted in inverted positions on glass slides and analyzed in confocal and multi-photon microscope (LSM 780; Carl Zeiss, Jena, Germany).

Statistical Analysis

The data were expressed as means ± SE. The Student's t test was used to compare control and treated groups, and when appropriate, a one-way analysis of variance followed by Tukey tests was used for multiple comparisons. P < .05 was used to indicate statistical significance. The statistical analysis was performed using the software package, GraphPad Prism (Prism 5.01; GraphPad Software, La Jolla, CA).

Results

Notch Signaling Is Upregulated after MAPK Activation

We asked whether activation of MAPK signaling could regulate the Notch pathway and we used a system with conditional activation of the RET/PTC3 or BRAFT1799A oncogenes by doxycycline in a normal rat thyroid cell line (PCCL3), designated PTC3-5 and PC-BRAF, respectively. The induction of RET/PTC3 or BRAFT1799A oncogenes by doxycycline was confirmed by Western blot as we observed a dose-dependent increase in ERK phosphorylation (pERK), a downstream component of MAPK signaling. Notch1 mRNA and protein levels were increased after RET/PTC3 or BRAFT1799A activation (Figure 1, A and B). The time course analysis revealed that Notch1 mRNA levels increased after 24 hours, with extended up-regulation 72 hours after oncogene induction in the PTC3-5 cell line (Figure 1C). During BRAFT1799A activation, up-regulation of Notch1 mRNA levels was also observed at 24 and 48 hours post-treatment. Higher Notch1 protein expression was showed after oncogene activation in PTC3-5 and PC-BRAF cell lines (Figure 1D). In addition, BRAFT1799A oncogene induction activates CBF1 luciferase reporter, suggesting the canonical activation of Notch signaling by MAPK pathway (Figure 1E). In PTC3-5 and PC-BRAF cell lines, oncogene activation increased HES1 mRNA levels (a downstream target gene of Notch signaling) after 48 and 72 hours, respectively, confirming the activation of Notch signaling by the RET/PTC3 and BRAFT1799A oncogenes (Figure 1F). Moreover, gene expression of the Notch signaling ligand, Jag1, was also upregulated after the activation of both oncogenes (Figure 1F), suggesting a potential mechanism for the up-regulation of Notch signaling by the MAPK pathway. To assess the influence of Notch signaling in a normal follicular thyroid cell proliferation, we used isogenic cell lines with ectopic expression of Notch1 intracellular domain (PC-NOTCH1). The growth curve analysis showed no modulation in PC-NOTCH1 cell growth (Figure W1).

Figure 1.

Figure 1

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RET/PTC3 and BRAFT1799A inducible system activates MAPK signaling pathway and increases Notch1 mRNA and protein expression. (A) PTC3-5 and PC-BRAF doxycycline-inducible cells were treated with the indicated concentration of doxycycline for 72 hours and real-time PCR was performed to determine Notch1 gene expression levels. mRNA levels are expressed as the fold change relative to the control group. (B) PTC3-5 and PC-BRAF cells were treated with the indicated concentration of doxycycline for 72 hours and Western blot of total protein lysates was carried out for Notch1 (115 kDa), phospho-ERK (pERK, 42 and 44 kDa), total ERK (42 and 44 kDa), and α-actin (42 kDa). (C) PTC3-5 and PC-BRAF doxycycline-inducible cells were treated with 1 µg/ml doxycycline for the indicated time periods and real-time PCR was performed to determine Notch1 gene expression levels. mRNA levels were expressed as the fold change relative to the control group. (D) PTC3-5 and PC-BRAF doxycycline-inducible cells were treated as indicated in C and Western blot of total protein lysates was performed. (E) CBF1 activity was measured in PC-BRAF cell transfected with the 4x CBF-Luc reporter plasmid containing four CBF1-responsive elements. Luciferase activity was measured 72 hours after BRAFT1799A oncogene induction with 1 µg/ml doxycycline. (F) PTC3-5 and PC-BRAF doxycycline-inducible cells were treated as indicated in C and real-time PCR was performed to determine Hes1 and Jag1 gene expression levels. mRNA levels were expressed as the fold change relative to the control group. Data are expressed as the means ± SE (n =3). *P < .05 versus control; **P < .01 versus control; ***P < .001 versus control.

To test whether Notch1 is modulated in vivo, we used transgenic mice Tg-BRAF, which harbor the BRAFT1799A oncogene under the control of the thyroglobulin promoter, driving the oncogene activation only in thyroid tissue [28]. Immunohistochemistry analysis of Notch1 protein expression in thyroid glands obtained from wild-type animals showed normal thyroid tissue architecture, with positive and negative staining for Notch1 in cells within the same follicle. In contrast, Tg-BRAF mice showed disrupted thyroid architecture, no evident colloid material, and strong positive staining for Notch1 in follicular and stromal cells (Figure 2A). In human PTC samples, 64% (9/14) showed elevated levels of NOTCH1 gene expression (Figure 2B). When NOTCH1 protein expression was analyzed by Western blot, non-tumor paired thyroid samples exhibited heterogeneous levels. However, PTC samples exhibited high levels of NOTCH1 protein expression when compared with nontumor samples (Figure 2C).

Figure 2.

Figure 2

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Notch1 expression is upregulated in a PTC mouse model and in human PTC samples. (A) Representative photomicrographs of immunostaining for Notch1 in wild-type and Tg-BRAF mouse thyroid gland tissue sections. Notch1 staining is shown in brown, and Gill's hematoxylin was used as a counterstain. The inset depicts the negative control. (B) NOTCH1 mRNA levels from human PTC and nontumor paired thyroid tissue samples were analyzed using real-time PCR. (C) NOTCH1 protein expression was analyzed by Western blot using antibodies against NOTCH1 (115 kDa) and α-actin (42 kDa) in human PTC and nontumor. N, nontumor sample.

Suppression of MAPK Signaling Pathway Decreases Notch Signaling

To establish whether Notch signaling activation in thyroid tumors is dependent on the MAPK pathway, the TPC-1 and BCPAP, human PTC cell lines harboring RET/PTC1 and BRAFT1799A, respectively, were treated with MEK inhibitors and NOTCH1 and HES1 mRNA levels were examined. BCPAP cell line was also treated with PLX4032, a specific inhibitor of mutated BRAFV600E. MAPK pathway inhibition was confirmed by markedly decreased pERK levels (Figure W2). NOTCH1 and HES1 mRNA levels were decreased after 48 hours of MAPK inhibition in the TPC-1 cell line (Figure 3). However, no significant changes in NOTCH1 or HES1 mRNA levels were observed after MAPK signaling inhibition in the BCPAP cell line (Figure 3).

Figure 3.

Figure 3

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Inhibition of the MAPK pathway decreases NOTCH1 and HES1 gene expression in TPC-1 cell line. TPC-1 and BCPAP cell lines were treated with the MEK inhibitors PD98059 (20 µM) and U0126 (10 µM), and BCPAP cell line was treated with PLX4032 (1 µM). After 48 hours (PD98059 and U0126 treatment) and 24 hours (PLX4032), real-time PCR was performed to determine NOTCH1 and HES1 gene expression levels. mRNA levels are expressed as the fold change relative to the control. Data are expressed as the means ± SE (n = 3). **P < .01 versus control; ***P < .001 versus control.

Notch Signaling Inhibition Reduces Proliferation of PTC Cell Lines

To assess the influence of Notch signaling in the proliferation of PTC cell lines, we used small-interfering RNA against NOTCH1. A reduction of NOTCH1 protein levels by 76% and 52% was observed after 24 and 48 hours of siRNA transfection (30 nM), respectively (Figure 4A). Silencing of NOTCH1 decreased cell proliferation and augmented apoptosis in TPC-1 cell line (Figure 4, B and C). We then used γ-secretase inhibitor (GSI) to pharmacologically deplete Notch signaling in TPC-1 and BCPAP cells. Immunofluorescence of Notch1 and gene expression analysis of the Notch target gene HES1 confirm the inhibition of Notch signaling by GSI, observed by NOTCH1 full-length accumulation at the cell membrane in TPC-1 cells (Figure 5A) and by a reduction in HES1 mRNA levels (Figure 5B). We observed a dose-dependent reduction of cell viability in PTC cell lines after treatment with GSI for 48 hours (Figure 5C). In addition, the treatment of PTC cell lines with GSI significantly decreased the cellular proliferation rate of both cell lines when compared to untreated cells (Figure 5D).

Figure 4.

Figure 4

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NOTCH1 gene silence regulates PTC cell proliferation. (A) TPC-1 cell line was transfected with siRNA targeting NOTCH1 (siN1) or siRNA targeting EGFP (siEGFP) with the indicated concentration, and 48 hours after transfection, total protein lysates were separated by SDS-PAGE and analyzed by Western blot for NOTCH1 (115 kDa) and α-tubulin (52 kDa). (B) TPC-1 cells were plated in six-well plates and transfected with siRNA targeting NOTCH1 (siN1; 30 nM) or siRNA targeting EGFP (siEGFP; 30 nM); the number of cells was determined over time. Data are expressed as the means ± SE (n = 3). (C) TPC-1 cell lines transfected with siRNA targeting NOTCH1 (siN1; 30 nM) or siRNA targeting EGFP (siEGFP; 30 nM), and after 96 hours, annexin V/propidium iodide staining followed by flow cytometry analysis was performed to quantify apoptosis (phosphatidylserine externalization). Data are expressed as the means ± SE (n =3). *P < .05.

Figure 5.

Figure 5

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GSI regulates PTC cell proliferation. (A) Double staining for Notch (green) and α-tubulin cytoskeleton expression (red) was performed in TPC-1 cells 24 hours after GSI (1 µM) treatment. Nucleus were stained with 4′,6-diamidino-2-phenylindole (blue). Scale bar, 50 µm. Arrows indicates NOTCH1 receptor accumulation in cell membrane. (B) TPC-1 and BCPAP cell lines were treated with GSI (1 µM) or vehicle solution (DMSO). After 48 hours, real-time PCR was performed to determine HES1 gene expression. mRNA levels were expressed as the fold change relative to the control group. Data are expressed as the means ± SE (n = 3). (C) TPC-1 and BCPAP cell lines were treated with the indicated concentration of GSI or vehicle solution (DMSO). After 48 hours, the percentage of viable cells was determined by MTT assay. (D) TPC-1 and BCPAP cells were plated in six-well plates and treated with GSI (1 µM) or vehicle solution (DMSO); the number of cells was determined over time. *P < .05 versus control; **P < .01 versus control; ***P < .001 versus control.

Pharmacological inhibition of Notch signaling modulates cell cycle-related genes in the TPC-1 cell line, increasing the mRNA levels of the cell cycle arrest-related genes CDKN1A and CDKN1B and decreasing the cell cycle progression-related genes CCND1, MCM6, CKS2, and MAD2L. Similar results were observed in the BCPAP cell line, except that significantly decreased CDKN1A mRNA levels were observed (Figure 6A). To determine the percentage of apoptotic cells, we treated PTC cell lines with GSI for 24 and 48 hours and double stained with FITC-conjugated annexin V and propidium iodide. Treatment of PTC cells with GSI increased apoptosis in a time-dependent manner (Figure 6B).

Figure 6.

Figure 6

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GSI regulates cell cycle-related genes and induces apoptosis in PTC cells. (A) TPC-1 and BCPAP cells were treated with GSI (1 µM) or vehicle solution (DMSO), and after 48 hours of treatment, real-time PCR was performed to quantify CCND1, CDKN1A, CDKN1B, MCM6, CKS2, and MAD2L gene expression. mRNA levels are expressed as the fold change relative to the control group. Data are expressed as the means ± SE (n = 3). (B) Annexin V/propidium iodide double staining followed by flow cytometry analysis was performed to quantify apoptosis (phosphatidylserine externalization) in TPC-1 and BCPAP cell lines treated with GSI (1 µM) or vehicle solution (DMSO) for the indicated time periods. Data are expressed as the means ± SE (n = 3). *P < .05 versus control; **P < .01 versus control; ***P < .001 versus control.

Inhibition of Notch Signaling Enhances the Growth Suppression Promoted by MEK Inhibitors in PTC Cells

Since it is well established that MAPK suppression regulates PTC cell growth, we assessed whether Notch signaling would be important for MAPK-mediated cell proliferation. Thus, we tested the influence of low-dose GSI treatment on the growth suppression induced by MAPK inhibition. As expected, after 48 hours an anti-proliferative effect was observed in PTC cells following the treatment with MEK inhibitors, with an especially marked response in BCPAP cells (Figure 7). The inhibition of Notch signaling by GSI produced no cumulative effect on cell viability when cells were concurrently treated with the PD98056 MEK1-specific inhibitor. However, the addition of GSI slightly enhanced the growth suppression induced by the MEK1/2 inhibitor U0126 in both PTC cell lines (Figure 7). These data indicate that the concomitant inhibition of Notch and MAPK signaling produce a cumulative effect on cell proliferation. No further effects were observed using higher concentrations of GSI (1 µM) along with the MEK inhibitors in either PTC cell line (data not shown).

Figure 7.

Figure 7

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GSI treatment enhances the growth suppression promoted by MAPK signaling in PTC cells. TPC-1 and BCPAP cell lines were treated with GSI (0.1 µm) and MEK inhibitor PD98059 (20 µM) or U0126 (10 µM), and after 48 hours, a cell viability assay was performed. Data are expressed as the means ± SE (n = 3). *P < .05 versus control; ***P < .001 versus control; #P < .01 versus U0126.

Discussion

Notch signaling has been implicated in the pathogenesis of different types of cancer [20]. Here, we demonstrate that Notch signaling is controlled by the MAPK pathway oncogenes, RET/PTC and BRAFT1799A, which are associated with thyroid cancer, and that inhibition of Notch signaling results in antiproliferative effect in PTC cells. Although Notch signaling has been shown to suppress tumor growth in cancer-derived keratinocytes, several lines of evidence indicate that it can play an oncogenic role in neurologic, hematological, and epithelial cancers, suggesting that overall result of Notch signaling is cell type dependent [19]. Up-regulation of NOTCH1 protein expression has been observed in pancreas and breast cancer [15,29]. In thyroid cancer, NOTCH1 expression differs among tumor histologic classification. In human anaplastic thyroid carcinoma, NOTCH1 gene expression is down-regulated [30]. By contrast, human PTCs, including microcarcinomas, classic and follicular variants, show higher expression of NOTCH1 when compared with normal thyroid tissues and peritumoral regions [31]. In addition, microarray analysis also indicates that several components of the Notch signaling pathway are upregulated in human PTC [21]. Corroborating these data, we observed an up-regulation of NOTCH1 expression in PTC samples derived from human and transgenic animals. Together, these data suggest that the Notch signaling pathway is dysregulated in PTC.

Genetic alterations in MAPK signaling oncogenes are implicated in the initial steps of PTC tumorigenesis [8,9,28]. By analyzing the influence of RET/PTC3 or BRAFT1799A in Notch signaling regulation, we demonstrated that both oncogenes implicated in thyroid tumorigenesis are involved in the up-regulation of Notch signaling. In nontumor thyroid cell lines, the MAPK pathway and Notch signaling cooperate to promote neoplasia. For instance, the MAPK pathway has been shown to regulate Jag1/Notch signaling and induces tumor angiogenesis in a head and neck squamous cell line [32] and tumorigenesis in pancreatic and lung cancer [29,33]. Although both RET/PTC and BRAF oncogenes can activate MAPK signaling, PTC oncogenes can elicit alternative pathways as well. RET/PTC and BRAFT1799A modulate different signaling pathways in PTC [34,35]. For instance, RET/PTC oncogene activates the β-catenin signaling pathway [36] and down-regulates let-7 microRNA [24], while BRAFT1799A induces nuclear factor-kappa B (NF-κB) signaling [37] and extracellular matrix remodeling [34]. Interestingly, blocking MAPK signaling through MEK inhibitors differentially regulates Notch signaling in PTC cell lines, TPC-1 (harboring RET/PTC1 rearrangement) and BCPAP (harboring BRAFT1799A mutation), indicating that different steps in the MAPK cascade control Notch expression.

Considerable attention has been focused on proliferative effects resulting from Notch signaling. In our model, we observed that Notch signaling inhibition regulates cell cycle regulatory genes that are tightly linked to cell cycle phase transition in PTC cells. In fact, Notch signaling directly regulates genes involved in cell cycle progression such as c-MYC [38], MCM2, MCM6 [39], and CCND1 [40] and cell cycle inhibition such as CDKN1A [41] and CDKN1B [42]. Moreover, we observed that inhibition of Notch signaling increased apoptosis in PTC cell lines. NOTCH1 gene silencing also decreased TPC-1 cell proliferation as observed in other epithelial cancer types, indicating the influence of NOTCH1 in cell proliferation [15,19].

Here, we show that genetic alterations in RET/PTC and BRAFT1799A in PTC lead to the activation of Notch signaling and contribute to tumor growth. Since Notch signaling components participates in tumor growth and are associated with poor prognosis in several types of cancer, including PTC, targeting Notch signaling could be a promising therapeutic approach to cancer treatment [43–46]. In conclusion, this work establishes a link between MAPK and Notch signaling pathways that control tumor growth in PTC. These data suggest that targeting Notch signaling may offer a potential adjuvant therapy for Notch-expressing PTC.

Supplementary Material

Supplementary Figures and Tables
tlo0602_0197SD1.pdf (163.6KB, pdf)

Acknowledgments

We thank the kind donation of cell line and mouse tissue by James Fagin and Jeff Knauf (Memorial Sloan Kettering Cancer Center, New York, NY), BCPAP cell line by Massimo Santoro (University Federico II of Naples, Naples, Italy), pBABE vectors by Anita Lal (University of California, San Francisco, CA), and 4x CBF1-Luc by Diane S. Hayward (Johns Hopkins University, Baltimore, MD). We thank the technical assistance of the Center of Facility and Support to Research (CEFAP), Institute of Biomedical Science and Kelly C. Saito for the immunofluorescence procedure.

Footnotes

1

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

2

This article refers to supplementary materials, which are designated by Table W1 and Figures W1 and W2 and are available online at www.transonc.com.

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