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Division of Medical Sciences, Institute of Biomedical Research, University of Birmingham, Birmingham B15 2TT, United Kingdom
Address all correspondence and requests for reprints to: C. J. McCabe, Division of Medical Sciences, Institute of Biomedical Research, University of Birmingham, Birmingham B15 2TT, United Kingdom. E-mail: mccabcjz{at}bham.ac.uk.
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Abstract |
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Objective: Our objective was to test whether VEGF, ID3, and KDR confer a PTTG-mediated effect on thyroid cell growth.
Design: Gene expression, MAPK stimulation, and cell proliferation were assessed in follicular thyroid cancer FTC133 cells. Gene expression and clinical associations were determined in 21 normal and 38 tumorous thyroid specimens (nine follicular and 29 papillary).
Results: ID3 correlated with VEGF mRNA expression in our series of thyroid cancers, which also showed up-regulated KDR mRNA. Stimulation of FTC133 cells with exogenous VEGF enhanced ID3 expression, which could be abrogated by the KDR-specific inhibitor ZM323881, suggesting that VEGF regulation of ID3 is KDR dependent. PTTG significantly correlated with KDR mRNA expression in our thyroid cancer cohort and up-regulated KDR and VEGF expression in FTC133 cells. Finally, cells transfected with PTTG demonstrated increased cell proliferation and phosphorylation of MAPK, which was abrogated by ZM323881.
Conclusions: We report the presence of a VEGF/KDR/ID3-dependent autocrine pathway in FTC133 thyroid cells. By up-regulating both VEGF and KDR expression, we propose a novel PTTG-mediated proliferative pathway that may be critical to thyroid cancer growth and progression.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Increased expression of pituitary tumor transforming gene (PTTG), a protooncogene (4), has been demonstrated in many cancer cell lines and human tumors compared with their normal counterparts (5, 6, 7, 8, 9, 10) and has been shown to play an important role in development of thyroid and other tumors (7, 11, 12). PTTG is a multifunctional protein (13, 14, 15, 16) and was initially demonstrated to promote angiogenesis via the up-regulation of fibroblast growth factor 2 (16). Subsequently, our group reported VEGF to also be up-regulated by PTTG (11). Furthermore, we observed a strong correlation between PTTG expression and KDR expression in pituitary tumors, suggesting a potential relationship between the function of these genes (11). More recently, we reported that PTTG regulates a number of pro- and antiangiogenic genes in thyroid cells, among them inhibitor of DNA-binding 3 (ID3).
VEGF receptors were initially believed to be expressed exclusively by endothelial cells. However, a growing number of studies have demonstrated expression of KDR and/or VEGFR1 in nonendothelial cells including ovarian, prostate, and pancreatic carcinoma cells, suggesting the existence of a potential autocrine stimulatory pathway (17, 18, 19, 20). VEGFR1 has been shown to be expressed in rat thyroid FRTL-5 cells (21). We examined the expression of KDR in a series of ex vivo human thyroid cancer specimens. To demonstrate receptor functionality, we assessed KDR-dependent MAPK intracellular signaling in response to exogenous VEGF. VEGF stimulated thyroid cell proliferation via a KDR-dependent pathway, with our data suggesting that up-regulation of the transcription factor ID3 may be involved in this autocrine mechanism. Furthermore, we demonstrated that PTTG up-regulates KDR expression and VEGF secretion in FTC133 follicular thyroid cancer cells, indicating that PTTG may play a critical role in thyroid tumorigenesis by promoting a VEGF/KDR/ID3 mitogenic autocrine mechanism in human thyroid cells.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Thyroid tumor tissues were obtained at the time of surgery from a consecutive series of patients undergoing thyroidectomy for thyroid cancer (n = 38; nine follicular and 29 papillary carcinomas). Twenty-one samples of histologically normal thyroid tissue were also obtained from patients undergoing laryngectomy for laryngeal cancer or from the thyroid lobe contralateral to the tumor; no normal thyroid specimen had any histological evidence of tumor invasion or small occult primary thyroid tumors. All of these tissues were excess to pathological requirements and were given with the patient’s written informed consent in accordance with local research ethics committee standards. Tissues were immediately snap frozen and stored at –80 C. Clinical and pathological details (tumor type, tumor-node-metastasis classification, and patient sex and age) including status during follow-up (mean duration from surgery, 31 months; range, 5–68 months) were available for 35 of the 38 differentiated thyroid cancers investigated.
Cell lines, plasmids, and transfections
Human follicular thyroid cancer FTC133 cells (Stratagene, Amsterdam, The Netherlands) were grown in Dulbecco’s medium NUT F12 (Ham’s), supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin (Life Technologies, Inc., Grand Island, NY). Before transfection experiments, cells were washed in PBS. Cells were transfected using Fugene-6 reagent (Promega Corp., Madison, WI), according to the manufacturer’s instructions but with an optimized ratio of 3 µl/µg plasmid DNA. pCI-neo-PTTG, which housed the full-length human PTTG cDNA, was kindly provided by Prof. Shlomo Melmed (University of California School of Medicine, Los Angeles, CA) (22). Transfection efficiency was assessed by using ß-galactosidase staining. Cells were harvested 48 h after transfection.
Experiments involving recombinant human VEGF (ab9571; Abcam, Cambridge, UK) and the selective KDR inhibitor ZM323881 (5-{[7-(benzyloxy) quinazolin-4-yl]amino}-4-fluoro-2-methylphenol) (23, 24) (Calbiochem, San Diego, CA) were performed in serum-free medium (SFM), unless otherwise specified. Cells were initially seeded onto plates and allowed to settle in 10% fetal bovine serum for 12 h, and then the medium was replaced with SFM for 12 h. Before addition of VEGF, the medium was replaced with fresh SFM. In relevant experiments, the KDR inhibitor ZM323881 was added (final concentration, 30 nM) 1 h before addition of VEGF. Cells were then incubated for 48 h.
RNA extraction and RT
Total RNA extraction from thyroid specimens and cell lines was performed with Trizol reagent (Sigma-Aldrich, Dorset, UK) according to the manufacturer’s instructions. RNA was reverse transcribed using avian myeloblastosis virus reverse transcriptase (Promega) in a total reaction volume of 20 µl, with 1 µg thyroid total RNA and 30 pmol random hexamer primers.
Quantitative PCR
Expression of specific mRNAs was determined using the ABI PRISM 7700 Sequence Detection System, as described previously (11, 25). Briefly, RT-PCR was carried out in 25-µl volumes on 96-well plates, in a reaction buffer containing 1x TaqMan Universal PCR Master Mix, 180 nM TaqMan probe, and 900 nM primers. PTTG and KDR reactions were multiplexed with a preoptimized control probe for ribosomal 18S RNA (PE Biosystems, Warrington, UK), enabling data to be expressed in relation to an internal reference, to allow for differences in RT efficiency. PTTG and KDR primers and probe sequences were as described previously (11, 25). Applied Biosystems (Warrington, UK) assay-on-demand primers and probe for ID3 were used (Hs00171409_m1). Validation studies were carried out to confirm parallel changes in target and housekeeping gene amplification across a range of RNA concentrations. According to the manufacturer’s guidelines, data were expressed as Ct values (the cycle number at which logarithmic PCR plots cross a calculated threshold line) and used to determine 
Ct values and, subsequently, fold changes in gene expression. All statistics were performed with Ct values. Measurements were carried out a minimum of three times for each sample. Reactions were 50 C for 2 min, 95 C for 10 min, and then 40 cycles of 95 C for 15 sec and 60 C for 1 min.
Western blot analysis
Protein samples from thyroid tissues and cultured cells were prepared in lysis buffer (100 mmol/liter sodium chloride, 0.1% Triton X-100, and 50 mmol/liter Tris, pH 8.3) containing enzyme inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.3 µM aprotinin, and 0.4 mM leupeptin) and denatured (2 min at 100 C) in loading buffer. Protein concentration was measured by the Bradford assay with BSA as standard.
PTTG Western blot analyses were performed as described previously (11, 25). For other genes, soluble proteins were separated by electrophoresis in 12.5% SDS-PAGE gels, transferred to polyvinylidene fluoride membranes, and incubated in 5% nonfat milk in PBS with 0.1% Tween, followed by incubation overnight at 4 C with p42/44 MAPK polyclonal antibody (Cell Signaling, Beverly, MA) at a 1:1000 concentration, phospho-p42/44 MAPK monoclonal antibody at a 1:4000 concentration (Cell Signaling), or phospho-KDR monoclonal antibody (kindly provided by Prof. Gatter, Oxford University, Oxford, UK) at a 1:100 concentration. After washing in PBS plus 0.1% Tween, blots were incubated with appropriate secondary antibodies conjugated to horseradish peroxidase. After additional washes, antigen-antibody complexes were visualized by the ECL chemiluminescence detection system on Hyperfilm ECL (Amersham Biosciences, Little Chalfont, UK). Actin expression (monoclonal anti-ß-actin clone AC-15 used at 1:10,000; Sigma-Aldrich) was also determined to control for differences in protein loading between different groups.
Cell proliferation assay
Cells (1 x 104/well) were plated onto 96-well plates and incubated in medium supplemented with 10% FBS for 12 h. Thereafter, cells were incubated in SFM for 12 h, fresh SFM replaced and then treated with recombinant VEGF and/or VEGFR2 inhibitor ZM323881 (30 nM) for a further 72 h. To each well, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (5 mg/ml) was then added for the final 3 h of the incubation. The crystals formed were dissolved by adding 200 µl dimethylsulfoxide to each well. The absorbance at 550 nm was measured on a Perkin-Elmer (Norwalk, CT) VICTOR3 Wallac 1420 microplate reader. The results were compared with respect to control values (untreated cells). MTT measurements were performed in triplicate with each experiment consisting of eight replicate wells.
VEGF ELISAs
Secreted VEGF in culture medium was measured using QuantiGlo-chemiluminescent ELISA according to the manufacturer’s instructions (R&D Systems, Abingdon, UK). Luminescence was measured on a Perkin-Elmer VICTOR3 Wallac 1420 microplate reader.
Statistical analyses
Statistical analyses were performed using Minitab version 14 software. Student’s t test was used for comparison between two groups of data. Correlation between pairs of mRNA results was examined using Pearson rank sum tests. Significance was taken as P value < 0.05.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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We have previously reported in separate investigations that PTTG up-regulates both VEGF (11) and ID3 (26). VEGF has been shown to up-regulate ID3 expression in endothelial cells, a critical mechanism in endothelial proliferation and angiogenesis (27). We thus investigated whether VEGF may similarly regulate ID3 in thyroid epithelial cells and whether this regulation was via KDR. First, we defined the relationship between VEGF and ID3 mRNA expression in human thyroid tumors and observed a highly significant positive correlation (R2 = 0.62; n = 38; P < 0.001) (Fig. 1A
). Furthermore, treatment of FTC133 cells with exogenous VEGF (20 ng/ml) led to significantly higher ID3 mRNA expression than untreated control cells (2.1-fold; n = 6; P < 0.001) (Fig. 1B). Up-regulation of ID3 by VEGF was abrogated by the addition of the specific KDR inhibitor ZM323881 (30 nM). Western blotting demonstrated consistent changes at the protein level (Fig. 1C). Taken together, our data suggest VEGF may up-regulate ID3 expression in FTC133 thyroid cells via KDR.
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We next investigated KDR expression in human ex vivo thyroid cancer specimens. KDR mRNA expression was 3.4-fold higher in differentiated thyroid cancers (n = 38) compared with normal thyroid tissue specimens (n = 21; P = 0.01) (Fig. 2A), although there was no significant difference in KDR expression between follicular (n = 9) and papillary (n = 29) tumors. When we examined the relationship between gene expression and clinical phenotype, we observed KDR mRNA expression in tumors associated with early recurrence to be significantly higher than both normal thyroid tissue (n = 21, P = 0.005) and tumors not displaying early recurrence (n = 32; P = 0.02). Also, tumors associated with nodal metastases at diagnosis demonstrated 8.4-fold higher KDR expression (n = 13; P = 0.001) than normal thyroid tissues (Fig. 2A) and significantly higher expression than node-negative tumors (n = 25; P = 0.013). Previously, in the same cohort of thyroid tissue specimens, we reported PTTG mRNA expression to be 4.1-fold increased in thyroid tumors compared with normal thyroid specimens (26).
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). PTTG up-regulates KDR and VEGF expression, and promotes autocrine and paracrine KDR-dependent mitogenic pathways in thyroid cells
Having observed a strong correlation between PTTG and KDR expression in thyroid cancers, we investigated whether PTTG regulates KDR expression in thyroid cells. KDR mRNA expression was significantly elevated in FTC133 cells transiently transfected with PTTG compared with vector-only (VO)-transfected control cells (2.2-fold; n = 7; P = 0.006) (Fig. 3A). Furthermore, Western blotting was performed using a monoclonal antibody for the ligand activated and phosphorylated KDR (pKDR) after transient overexpression of PTTG. Compared with VO, PTTG transfection resulted in increased pKDR expression (Fig. 3A). Consistent with previously studied nonthyroidal cell lines (11), FTC133 cells transfected with PTTG showed significantly higher VEGF mRNA expression (2.4-fold; n = 4; P < 0.001) (Fig. 3B) and, using ELISA, secreted significantly increased VEGF protein compared with VO-transfected control cells (VO, 26.9 ± 0.2 ng/ml; PTTG, 34.2 ± 0.2 ng/ml; n = 8; P < 0.001) (Fig. 3C).
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We examined phosphorylation of MAPK, a key downstream intracellular signaling molecule involved in the VEGF-induced mitogenic pathway (28). In keeping with raised KDR expression and VEGF secretion, and thus the potentiation of an autocrine pathway, we observed increased phosphorylation of MAPK in cells transfected with PTTG compared with VO-transfected control cells (Fig. 4A). Addition of 30 nM of the selective KDR inhibitor ZM323881 to PTTG-transfected cells abrogated this effect. To investigate the endogenous expression of KDR mRNA in FTC133 cells, TaqMan RT-PCR was carried out as well as in a thyroid endothelial cell line (29) as a positive control for KDR expression (Fig. 4B). In parallel, Western blotting studies revealed bands at 230, 150, and 70 kDa (Fig. 4B). The expected product at 230 kDa represents the fully glycosylated KDR protein within the outer cell membrane. Hence, FTC133 cells express KDR mRNA and protein, and pMAPK is up-regulated by PTTG, an effect abrogated by a selective KDR inhibitor.
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). Furthermore, when the selective KDR inhibitor ZM323881 was added to cells grown in the conditioned medium from PTTG-transfected cells, increased cell proliferation was not observed. Indeed, a significantly lower rate of proliferation was observed compared with cells grown in conditioned medium derived from VO-transfected cells (P < 0.001; n = 3) (Fig. 4C). VEGF stimulates FTC133 cell proliferation by activation of KDR-dependent MAPK mitogenic pathway
Based on the above evidence, we hypothesized that PTTG stimulation of FTC133 cell proliferation was mediated by VEGF acting via its receptor KDR. To test whether enhanced VEGF levels alone could recapitulate our PTTG findings, we treated FTC133 cells with exogenous VEGF. In response to 10–50 ng/ml doses of VEGF, we observed an increase in phosphorylation of MAPK in FTC133 cells (Fig. 5Ai). This effect appeared to be optimal by 20 ng/ml. Unphosphorylated MAPK expression was unaltered, indicating that pMAPK expression changes represented increased phosphorylation rather than translational effects. We also assessed the expression of pKDR in response to VEGF. pKDR was similarly induced by 10 and 20 ng/ml exogenous VEGF compared with vehicle (Fig. 5Aii).
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). In addition, use of the selective KDR inhibitor ZM323881 (30 nM) markedly suppressed VEGF’s mitogenic effect. Additionally, when we compared our proliferation data with ID3 mRNA expression in parallel experiments (see Figs. 1B and 5B), we observed a strong correlation between ID3 mRNA expression and cell proliferation (R2 = 0.81; n = 8; P = 0.002). Taken together, these data suggest that the high PTTG expression apparent in differentiated thyroid tumors may promote a novel autocrine mitogenic mechanism of tumor promotion by up-regulating the expression of both VEGF and its receptor KDR in thyroid cells. |
Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The rationale behind such therapy has been to inhibit VEGF-driven angiogenesis and thus inhibit cancer growth. However, more recently, against the previous notion that VEGF receptors were exclusive to endothelial cells, an increasing number of studies have demonstrated expression of functional VEGF receptors in nonendothelial cells (19, 33, 34, 35). This suggests the potential for VEGF stimulation of cancer cells through an autocrine pathway and that tumors exhibiting such a VEGF-KDR autocrine pathway may thus be more amenable to anti-VEGFR therapies. Indeed, recent work in murine models of follicular thyroid cancer demonstrated that the use of the joint KDR and epidermal growth factor receptor inhibitor NVP-AEE788 reduced FTC cell proliferation in vitro and thyroid tumor growth in vivo (36) and indicated a role for KDR inhibitors in the treatment of bone-metastatic thyroid tumors (37).
In the present study, we demonstrated that KDR expressed in FTC133 thyroid cells exhibited the proper functional response to VEGF binding by showing activation of the KDR-dependent downstream MAPK signaling pathway and KDR-dependent mitogenesis. Activation of KDR by VEGF triggers multiple downstream intracellular signaling pathways (38). Although we observed that both pKDR and the mitogenic ERK-MAPK pathway were stimulated by VEGF in FTC133 cells, it is unclear which genes are subsequently involved in promoting thyroid cell proliferation. Up-regulation of the transcriptional factor ID3 in human umbilical vein endothelial cells has been demonstrated to be important in the VEGF-dependent angiogenic process (27). More recently, we reported elevated ID3 expression in differentiated thyroid cancers (26) and that PTTG regulates ID3 mRNA and protein expression in thyroid cells in vitro. In the present study, we therefore examined ID3 and VEGF mRNA expression in a series of differentiated thyroid tumors and demonstrated a highly significant and strong correlation between expression of these genes. Also, treatment of FTC133 cells with exogenous VEGF was associated with significant up-regulation of ID3 expression compared with control cells. However, ID3 up-regulation in response to VEGF stimulation was abrogated with the addition of a KDR-specific inhibitor, suggesting that VEGF regulation of ID3 is KDR dependent. Furthermore, we observed a strong correlation between the rate of cell proliferation and ID3 expression in FTC133 cells in these experiments. Taken together, these data suggest that VEGF may be involved in an autocrine mitogenic pathway by activating its KDR receptor, which in turn up-regulates ID3 expression, a nuclear transcriptional factor known to have potent proliferative properties (39). One caveat to this is that although ZM323881 is a highly selective KDR inhibitor (23) and has been shown not to affect VEGFR1, epidermal growth factor receptor, platelet-derived growth factor receptor, or hepatocyte growth factor receptor pathways (24), it is possible that ZM323881 could influence other signal transduction pathways that would not have been detected in the present study.
Previously, we have reported higher KDR expression in pituitary tumors than in normal pituitary and a strong correlation between PTTG and KDR mRNA expression (11), suggesting a potential regulatory relationship between these two genes. We therefore examined this relationship in our series of thyroid cancers. We observed significantly higher KDR expression in thyroid cancers compared with normal thyroid tissue. KDR expression was also significantly higher in tumors displaying nodal spread and early recurrence compared with node-negative and nonrecurring tumors, respectively. One specific difficulty, however, lies in discriminating endothelial cell KDR from follicular cell KDR, given that angiogenesis may be increased in thyroid cancer. Previously, Vieira et al. (40) showed thyroid follicular cell expression in papillary and follicular thyroid cancers using immunostaining, although they did not attempt quantitation of staining intensity, only frequency of expression. Additional studies are therefore needed to define the potential clinical importance of KDR expression as a predictive marker in thyroid cancer. Despite this, our data are in keeping with other studies that show increased KDR expression in several human tumor types (11, 19, 41) and increased VEGFR1 in thyroid cancer (30). Furthermore, we demonstrated a highly significant association between PTTG and KDR expression.
To examine this potential relationship in more detail, we transfected FTC133 cells with PTTG and showed a significant up-regulation in KDR mRNA and protein expression compared with control cells. Also, as reported previously in other cell types (11), we demonstrated FTC133 cells overexpressing PTTG to be associated with significantly higher VEGF mRNA expression and to secrete a significantly increased amount of VEGF protein compared with VO-transfected control cells. In keeping with both a raised KDR expression and increased VEGF secretion, FTC133 cells expressing high levels of PTTG demonstrated significantly greater KDR-dependent MAPK activation than VO-transfected control cells. Furthermore, thyroid cells grown in conditioned medium from PTTG-transfected FTC133 cells demonstrated greater cell proliferation compared with cells grown in conditioned medium from untransfected cells and cells cultured in SFM. These findings suggest that elevated PTTG expression may promote an autocrine stimulatory pathway in thyroid cells by stimulating both VEGF secretion and KDR expression.
In summary, we have shown VEGF to stimulate both FTC133 cell proliferation and ID3 expression, a potent mitogenic transcriptional factor, via KDR activation. We also observed significantly higher KDR mRNA expression in thyroid cancers and that the highest KDR expression was found in tumors displaying early recurrence and nodal metastasis at diagnosis. Furthermore, PTTG overexpression was shown to increase both VEGF secretion and KDR expression in FTC133 cells. We propose, therefore, that the high PTTG expression observed in thyroid cancers may promote a VEGF/KDR/ID3 autocrine mitogenic pathway and suggest that this may be a critical mechanism in thyroid tumor progression.
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Footnotes |
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Disclosure statement: The authors have nothing to disclose.
First Published Online August 22, 2006
Abbreviations: ID3, Inhibitor of DNA binding-3; KDR, kinase insert domain receptor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; pKDR, phosphorylated KDR; PTTG, pituitary tumor transforming gene; SFM, serum-free medium; VEGF, vascular endothelial growth factor; VEGFR1, VEGR receptor type 1; VO, vector-only.
Received June 16, 2006.
Accepted August 14, 2006.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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