| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Division of Medical Sciences, Institute of Biomedical Research, University of Birmingham, Edgbaston, Birmingham B15 2TH, United Kingdom
Address all correspondence and requests for reprints to: Dr. Dae Kim, Division of Medical Sciences, Institute of Biomedical Research, University of Birmingham, Edgbaston, Birmingham B15 2TH, United Kingdom. E-mail: daekim72{at}yahoo.co.uk.
 |
Abstract |
|---|
 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
|---|
Objective: To investigate whether PTTG regulates other angiogenic genes in thyroid cells, we performed angiogenesis-specific cDNA arrays after PTTG transfection. Two of the genes [inhibitor of DNA binding-3 (ID3) and thrombospondin-1 (TSP-1)] which showed differential expression in primary thyroid cells were validated in vitro and in vivo.
Results: TSP-1 showed a 2.5-fold reduction and ID3 showed a 3.5-fold induction in expression in response to PTTG overexpression in vitro. Conversely, suppression of PTTG with small interfering RNA was associated with a 2-fold induction of TSP-1 and a 2.2-fold reduction in ID3 expression. When we examined TSP-1 and ID3 expression in 34 differentiated thyroid cancers, ID3 was significantly increased in tumors compared with normal thyroid tissue. Furthermore, ID3 expression was significantly higher in follicular thyroid tumors than in papillary tumors. Although mean TSP-1 expression was not altered in cancers compared with normal thyroids, we observed a significant independent association between TSP-1 expression and early tumor recurrence, with recurrent tumors demonstrating 4.2-fold lower TSP-1 expression than normal thyroid tissues.
Conclusion: We have identified ID3 and TSP-1 as two new downstream targets of PTTG in thyroid cancer. We propose that PTTG may promote angiogenesis by regulating the expression of multiple genes with both pro- and antiangiogenic properties and may thus be a key gene in triggering the angiogenic switch in thyroid tumorigenesis.
|
Introduction |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
Pituitary tumor-transforming gene (PTTG) is a protooncogene first identified from rat pituitary GH4 cells in 1997 (4). Increased expression of PTTG has been shown in many cancer cell lines and human tumors, including colorectal, breast, hemopoietic, and pituitary (5, 6, 7, 8, 9). We have previously reported significantly higher expression of PTTG and fibroblast growth factor-2 (FGF-2) in thyroid cancers compared with normal thyroid tissues and also showed that increased levels of PTTG and FGF-2 expression in thyroid cancers are independent predictors of early tumor recurrence (10). Others have demonstrated high PTTG expression in breast and colorectal cancers to predict recurrence as well as the presence of nodal and distant metastases (6, 9). PTTG is a multifunctional protein involved in several tumorigenic mechanisms, including angiogenesis (11). Using multiple assays, PTTG was shown to promote angiogenesis in vivo, with FGF-2 up-regulation an important pathway. Subsequently, we and others have reported that PTTG also up-regulates the angiogenic factor vascular endothelial growth factor (VEGF) in vitro (12).
Detailed analyses of the earlier functional studies suggested that other growth factors are likely to be involved in PTTG-dependent angiogenesis (11). To investigate this, we employed focused angiogenesis-specific cDNA arrays and identified multiple angiogenic genes that are differentially expressed in PTTG-transfected cells. Two of these genes, inhibitor of DNA binding-3 (ID3) and thrombospondin-1 (TSP-1), were chosen for additional validation in a series of 34 differentiated thyroid tumors. We show that both angiogenic promoter and angiogenic inhibitor genes demonstrate differential expression in response to elevated PTTG levels in thyroid cells, and we propose, therefore, that PTTG plays an important role in the angiogenic switch process in thyroid cancer.
|
Patients and Methods |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
Thyroid tumor tissues were obtained at the time of surgery from a consecutive series of patients undergoing thyroidectomy for thyroid cancer (n = 34; eight follicular and 26 papillary carcinomas). Eighteen samples of histologically normal thyroid tissue were obtained from patients undergoing laryngectomy for laryngeal cancer or from the thyroid lobe contralateral to the thyroid tumor; no normal thyroid specimen had any histological evidence of tumor invasion or the presence of small occult primary thyroid tumors. All these tissues were excess to pathological requirements and were donated 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. Full clinical and pathological data were recorded for each tumor. Early recurrence was defined as evidence on imaging of recurrent disease in the thyroid bed or elsewhere or rising serum thyroglobulin levels in association with TSH suppression during T4 therapy. Subsequently, tumor recurrence was confirmed histologically in three of the six cases, and in the remaining three by demonstrating regression of lesions on imaging and falling thyroglobulin levels after ablative radioiodine therapy.
Cell lines, plasmids, and transfections
Human thyroid cells were isolated and cultured as described by Eggo et al. (13). In brief, thyroid tissue obtained at surgery in accordance with local ethical guidelines was used. Multinodular goiter, removed to relieve tracheal compression, was trimmed of extraneous connective tissue and chopped finely with scalpel blades. After 3-h digestion at 37 C in 0.1% collagenase to release follicles, the material was repeatedly washed by centrifugation. Cells were plated at low density in Coon’s Modified Ham’s F-12 medium with TSH (0.3 nmol/liter; 300 mU/liter), insulin (160 nmol/liter), penicillin (100 µU/ml), streptomycin (100 µg/ml), and 1% bovine calf serum on six-well plates. At the first medium change, serum was omitted from the medium, and cells were cultured without serum thereafter. Medium was changed twice weekly. Cells were screened for their ability to take up and organify iodide, and those cultures that showed TSH-dependent iodide uptake and organification were used in these studies. We used these parameters to confirm that the cells expressed full differentiated function and were responsive to TSH. 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 (Invitrogen Life Technologies, Inc., Grand Island, NY).
pCI-neo-PTTG, which housed the full-length human PTTG cDNA, was provided by Dr. Shlomo Melmed (University of California School of Medicine, Los Angeles, CA) (14). The PTTG Src homology domain 3 (SH3) mutation, which disrupts the C-terminal SH3-interacting domain of PTTG, was recreated in the pCI-neo-PTTG vector using the GeneEditor System (Promega Corp., Madison, WI) according to the manufacturer’s instructions. The mutagenic primer sequence and the resulting amino acid changes have been previously detailed (15). Before transfection experiments, cells were washed in PBS. Cells were transfected using FuGene-6 reagent (Promega Corp.) according to the manufacturer’s instructions, but with an optimized ratio of 3 µl/µg plasmid DNA. Cells were harvested in 1 ml TRIzol reagent 48 h after transfection. Transfection efficiency was assessed by ß-galactosidase staining.
PTTG knockdown experiments were carried out with a PTTG-specific predesigned small interfering RNA (siRNA; ID no. 42168, Ambion, Inc., Huntingdon, UK) and negative control 1 siRNA (ID no. 4611; Ambion, Inc.). FTC133 cells were transfected using siPORT NeoFX transfection reagent (Ambion, Inc.) according to the manufacturer’s instructions. In brief, cells were trypsinized, suspended in culture medium, and mixed with siRNA (10–20 nM) and NeoFX transfection reagent (3 µl/ml medium) before plating and incubation for 48 h.
RNA extraction and RT
Total RNA extraction from thyroid specimens and cell lines was performed with TRIzol reagent (Sigma-Aldrich Corp., Dorset, UK). RNA extraction has been described previously in detail (10, 12). RNA was reverse transcribed using avian myeloblastosis virus reverse transcriptase (Promega Corp.) 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 (Applied Biosystems, Foster City, CA), as described previously (10, 12). Predesigned probe and primers were chosen from ABI Biosystems (Warrington, UK) for ID3 (ID no. Hs00171409_m1) and thrombospondin 1 (ID no. Hs00170236_m1). The PTTG primers and probe sequences used were described previously (10, 12). 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 (Eurogentec, Southampton, UK), 180 nm TaqMan probe, and 900 nM primers. Reactions were conducted at 50 C for 2 min, 95 C for 10 min, then 40 cycles of 95 C for 15 sec and 60 C for 1 min. All reactions were multiplexed with a preoptimized control probe for ribosomal RNA 18S (ABI Biosystems), enabling data to be normalized in relation to an internal reference, as is standard for quantitative RT-PCR analysis, to allow for variations in RNA concentration, transfection efficiency, and other technical variables. 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 were used to determine 
Ct values (Ct = Ct of the target gene minus Ct of the housekeeping gene 18S). The fold change in mRNA expression in an experimental group compared with the control was calculated using the equation: fold change = 2–(DCt of experimental group – DCT of control group) or 2–Ct. All statistics were performed with Ct values. Measurements were carried out a minimum of three times for each sample.
cDNA microarray analysis
Total RNA was extracted using TRIzol (Molecular Research Center, Inc., Cincinnati, OH) and was cleaned using the QIAGEN RNeasy kit (QIAGEN, Crawley, UK). A Nonradioactive Angiogenesis GEArray Q-series Kit (SuperArray, Inc., Bethesda, MD) consisting of nylon arrays tetra-spotted with 96 angiogenic genes was used to compare gene expression in wild-type (WT)-PTTG transfected primary thyroid cells with cells transfected with empty expression vector alone (VO-control). Briefly, 5 mg total RNA was used as a template for biotinylated cDNA probe synthesis. RNA was reverse transcribed by gene-specific primers with biotin-16-deoxy-UTP. Biotinylated cDNA probes were denatured and hybridized to the array membranes. The GEArray membranes were then washed and blocked with GEA blocking solution, and incubated with alkaline phosphatase-conjugated streptavidin. The hybridized biotinylated probes were detected by the chemiluminescent method using the alkaline phosphatase substrate, CDP-Star (SuperArray, Frederick, MD).
The expression levels of different genes were first analyzed using ScanAlyse (Michael Eisen Software, Stanford, CA). GEArray (Super-Array) analyzer software was then used to match the raw data with the specific microarray gene table list to allow background subtraction and normalization of the raw data. A similar method of analysis of the 96-gene arrays was previously used widely (16, 17, 18). Briefly, the local background correction method was used, where each expression value has an individual subtraction based on the area outside grid capture, but within the spot cell area. The expression of each gene was then normalized to the median values of the housekeeping genes ß-actin and glyceraldehyde-3-phosphate dehydrogenase to allow valid comparison with duplicate arrays.
Cell transfection experiments were carried out in triplicate, and the total mRNAs were pooled before probe synthesis. Thereafter, each array experiment was performed in duplicate. The values for gene expression were averaged for the duplicate array experiments. Those genes that demonstrated more than a ±2.0 average fold differential expression in PTTG-transfected cells compared with VO-transfected control cells from duplicate array experiments were tabulated as significant genes of interest.
Western blot analysis
Proteins were prepared from thyroid tissues 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 phenylmethylsulfonylfluoride, 0.3 µM aprotinin, and 0.4 mM leupeptin) and denatured (2 min, 100 C) in loading buffer. The protein concentration before loading was measured using the Bradford assay with BSA as standard.
Western blot analyses were performed as described previously (10, 12). Briefly, soluble proteins were separated by electrophoresis in 12.5% sodium dodecyl sulfate-polyacrylamide gels, transferred to polyvinylidene fluoride membranes, incubated in 5% nonfat milk in PBS with 0.1% Tween, followed by incubation overnight at 4 C with a monoclonal PTTG antibody at a 1:1,000 concentration (NeoMarker, Fremont, CA), ID3 polyclonal antibody at a 1:200 concentration (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and TSP-1 polyclonal antibody (Santa Cruz Biotechnology, Inc.) at a 1:500 concentration. After washing in PBS plus 0.1% Tween, blots were incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase overnight at 4 C. 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 Corp.) was also determined to control for differences in protein loading between different groups.
Statistical analyses
Statistical analyses were performed using Minitab version 14 software (Minitab Ltd., Coventry, UK). Student’s t test was used for comparisons between two groups of data. Correlation between pairs of mRNA results was examined using Pearson rank-sum tests. Significance was taken as P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
Using the commercial GEArray analysis suite, we identified 28 angiogenesis-related genes to be differentially regulated (>2-fold up- or down-regulated in duplicate array experiments) in response to increased PTTG expression in transiently transfected primary thyroid cells (Table 1
). A total of 18 genes were up-regulated, and 10 genes were down-regulated in PTTG-transfected cells compared with VO-control cells.
|
It was beyond the scope of the present studies to investigate mRNA and protein expression of all 28 genes showing altered expression on our arrays. Therefore, to examine the validity of our finding, and specifically to test our hypothesis that PTTG regulates both pro- and antiangiogenic genes, we chose to study one positive and one negative regulator of angiogenesis, focusing on genes we believed would be mechanistically relevant to PTTG’s role in thyroid cancer angiogenesis. We thus selected the angiogenic promoter ID3, which has been shown to play a critical role in cell proliferation (19) and precursor endothelial cell recruitment (20), and the angiogenic inhibitor TSP-1, which has been demonstrated to predict poor survival (21) and represent a marker of tumor aggressiveness in thyroid cancer (22).
Using the same total mRNAs that were used to probe the array membranes, TaqMan RT-PCR analysis confirmed up-regulation of ID3 mRNA expression (62%; n = 3; P = 0.0053; Fig. 1A) and down-regulation of TSP-1 expression (86%; n = 3; P < 0.001; Fig. 1B) in PTTG-transfected primary thyroid cells. Additional transfection experiments using additional primary thyroid cells demonstrated these observations to be reproducible [ID3 was up-regulated 61% (n = 6; P = 0.005) and TSP was down-regulated 54% (n = 6; P = 0.012) by PTTG over-expression; Fig. 2, A and B]. Finally, Western blotting demonstrated changes in protein expression in keeping with the changes apparent at the mRNA level (Fig. 2, A and B).
|
|
The proline-rich putative SH3-binding domain of PTTG has been shown to mediate its stimulation of FGF-2 and VEGF expression (15). We investigated whether this important region of the PTTG protein is essential in regulating ID3 and TSP-1 expression. Compared with the VO-control, WT-PTTG significantly stimulated ID3 expression (1.94-fold; n = 5; P = 0.003). However, when the SH3 mutant form of PTTG, which shows identical stability to WT-PTTG (15), was transfected into primary thyroid cells, the stimulatory effect was significantly reduced (1.32-fold; n = 5; Fig. 2C
). Consistent protein expression data were obtained with Western blotting. However, TSP-1 expression was not significantly affected by the SH3 mutant construct in that both overexpression of WT-PTTG and SH3 mutant PTTG similarly suppressed TSP-1 mRNA expression in primary thyroid cells compared with VO-control transfected cells [WT-PTTG, 0.46-fold (n = 4; P = 0.012); SH3 mutant, 0.42-fold (n = 5; P = 0.039); Fig. 2D].
PTTG regulates ID3 and TSP-1 expression in FTC133 human follicular cancer cells
In addition to primary thyroid cells, we were able to confirm down-regulation of TSP-1 (0.45-fold; n = 3; P = 0.002) and up-regulation of ID3 (2.2-fold; n = 3; P < 0.001) mRNA expression in FTC133 human follicular cancer cells (Fig. 3A). Furthermore, we used PTTG-targeted siRNA to suppress PTTG expression and examined ID3 and TSP-1 levels in these cells. As demonstrated with many other cancer cell lines (5, 6, 7, 8), FTC133 cells showed high constitutive PTTG expression, particularly in comparison with primary thyroid cells (data not shown). Suppression of PTTG expression using 20 nM siRNA (Fig. 3B) was associated with up-regulation of TSP-1 (2.09-fold; n = 4; P < 0.001) and down-regulation of ID3 mRNA expression (0.45-fold; n = 4; P = 0.0014; Fig. 3C). Western blotting demonstrated changes in protein expression in keeping with the changes apparent at the mRNA level (Fig. 3C).
|
PTTG mRNA expression was 4.1-fold higher in differentiated thyroid tumors than in normal thyroid specimens (Fig. 4A). There was no significant difference in PTTG expression between follicular and papillary tumor subtypes. Having shown high PTTG expression to be associated with increased expression of ID3 and reduced TSP-1 expression in vitro, we next investigated whether in vivo expression of ID3 and TSP-1 in thyroid tumors was consistent with our in vitro findings.
|
). Clinical and pathological details [tumor type, TNM (tumor/node/metastases) classification, patient sex, and patient age], including status during follow-up (mean duration from surgery, 31 months; range, 5–68 months), were available for 32 of the 34 differentiated thyroid cancers investigated. ID3 expression in papillary tumors was 3.5-fold (n = 26; P = 0.016) higher than that in normal thyroid tissues, whereas follicular tumors demonstrated a 15.7-fold (n = 8; P < 0.001) increase in ID3 expression compared with normal tissues. ID3 expression in follicular tumors was significantly higher than that in papillary thyroid tumors (P = 0.013; Fig. 4B). Also, in nine of 11 patient-matched, normal tumor thyroid sample pairs, ID3 mRNA expression was elevated in cancer tissue compared with normal tissue from the same patient (Fig. 4C). Consistent with this, and with our in vitro findings, eight of 11 cancers demonstrated a parallel association between PTTG and ID3 gene expression (Fig. 4, C and D).
In contrast to this, there was no significant difference in mean TSP-1 expression between tumors (n = 34) and normal specimens (n = 18), and there was no difference in TSP-1 expression between papillary and follicular thyroid neoplasms. Indeed, analysis of the same 11 patient-matched normal and thyroid cancer tissue pairs revealed highly variable TSP-1 expression (Fig. 4, D and E). Seven of 11 cancers showed reduced TSP-1 expression, whereas the remaining four were associated with elevated TSP-1 expression. Supportive of our in vitro findings, however, eight of 11 cancers demonstrated an inverse association between PTTG and TSP-1 expression, with elevated PTTG expression associated with low TSP-1 expression, and low PTTG apparent in cancers expressing elevated TSP-1. Comparing mRNA expression values for PTTG and TSP-1 overall, we observed a highly significant negative correlation (r2 = 0.55; P = 0.0036) between the two genes.
Associations between gene expression and clinico-pathological parameters
When we examined the relationship between gene expression and patient data, we observed a significant association between ID3 expression and age at diagnosis. Compared with normal thyroid tissues, ID3 expression was up-regulated 19.0-fold in tumors from those patients presenting over the age of 45 yr (n = 13) and 2.0-fold in tumors from patients (n = 19) presenting below this age (P = 0.002; Fig. 5A). Multiple linear regression analysis (taking into account the known prognostic indicators of age, gender, and tumor type) confirmed independent associations between ID3 expression and age at diagnosis (r2 = 0.32; P < 0.001) as well as tumor type (r2 = 0.12; P = 0.049). However, TSP-1 expression showed a significant association with early tumor recurrence (six tumors recurred within 3 yr of follow-up). Tumors associated with early recurrence showed 4.2-fold lower TSP-1 expression than normal thyroid tissue (P = 0.04; Fig. 5B). Finally, multiple linear regression analysis showed independent association between TSP-1 expression and recurrence, taking into account the known prognostic indicators of age, gender, and tumor type (r2 = 0.16; P = 0.023).
|
|
Discussion |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
PTTG is a multifunctional protein (11, 23, 24) that plays an important role in thyroid and other tumor development (12, 25). PTTG was initially shown to promote angiogenesis via the up-regulation of FGF-2 (11). Subsequently, our group reported VEGF to be additionally up-regulated by PTTG (12). Earlier functional studies had suggested that other angiogenic factors were likely to be involved in this PTTG-driven angiogenic process (11). To investigate the specific angiogenic factors that may be regulated by PTTG and the overall extent of its proangiogenic effects, we employed a focused microarray strategy. Our present study suggests that PTTG overexpression in primary thyroid cells is associated with both the enhanced expression of angiogenic stimulators and the repression of angiogenic inhibitors. The precise role played by each of these angiogenic factors awaits additional investigation.
Of the multiple genes shown to be differentially expressed, it is likely that a small number of angiogenic genes are critical to PTTG’s direct promotion of angiogenesis, whereas others may be involved downstream of these key genes. However, in tissues or disorders in which PTTG expression is not significantly elevated, such as Graves’ disease, where there is nonetheless evidence for increased angiogenesis and perfusion, other important proangiogenic factors are likely to be involved.
To prioritize, we chose to study one positive and one negative regulator of angiogenesis, focusing on genes we believed would best advance our specific understanding of PTTG’s role in thyroid cancer angiogenesis. We thus selected the angiogenic promoter ID3 and the angiogenic inhibitor TSP-1. PTTG has been shown to up-regulate VEGF expression (12). It has been reported that VEGF may up-regulate ID3 expression (26), an angiogenic gene believed to play a critical role in cell proliferation (19) and precursor endothelial cell recruitment (20). Given that ID3 was differentially expressed in thyroid cells expressing high PTTG levels, we aimed to validate our findings with a view to clarifying the interactions among PTTG, VEGF, and ID3. TSP-1 was chosen because it is secreted in significant quantities by thyroid follicular cells, second only to thyroglobulin, and is believed to play a role in epithelial cell growth and cell-cell adhesion (27). TSP-1 has also been shown to predict poor survival (21) and represent a marker of tumor aggressiveness in thyroid cancer (22).
ID proteins are believed to play an important role in the initiation and progression of several tumor types (28, 29, 30). Deregulation in expression of ID protein has been shown to be important in the control of cell growth, proliferation, angiogenesis, and invasiveness (31). There is tissue-specific expression of ID proteins, with ID1 and ID3 genes closely coexpressed during many processes, including angiogenesis (19, 26). We have shown in the current study that PTTG regulates the expression of ID3 mRNA and protein in primary human thyroid cells. The mechanisms underlying this are not known, but may be both direct and indirect. We were able to significantly reduce this effect using an SH3 mutant form of PTTG, suggesting that the SH3-binding domain of the PTTG protein is important for its influence on ID3 expression. This region of the protein has been shown previously to be involved in up-regulating both VEGF and FGF-2 (12, 15). Proteins containing SH3 domains are integral to protein-protein interactions and are intimately involved in regulating intracellular pathways controlling gene expression and DNA synthesis (32). It is thus likely that PTTG acts via such regulatory intermediary proteins with SH3 domains to regulate ID3 expression. Indeed, protein kinase A and C mitogenic pathways, which employ SH3 protein signaling and are activated by many upstream triggers, have been shown to up-regulate ID gene expression (33). The C-terminal portion of PTTG contains a DNA-binding domain that has been demonstrated to be involved in directly stimulating the c-myc promoter (34). It is also possible, therefore, that a direct interaction with the ID3 gene may contribute in part to PTTG’s regulatory effect.
We have also shown, using quantitative RT-PCR analysis of a large differentiated thyroid tumor cohort, that ID3 expression is significantly higher in differentiated thyroid cancers compared with normal thyroid tissue. Furthermore, in nine of 11 patient-matched, normal thyroid-thyroid tumor sample pairs, ID3 expression was significantly higher in cancer specimens. Our results are consistent with ID gene expression reported previously in numerous other tumor types (19, 35) and are in keeping with a recent study that showed higher ID1 gene expression in 46 thyroid cancers compared with normal thyroid tissues (30). However, the regulation of ID3 expression is mutifactorial (33), and alternative regulatory factors that are more potent than the effects of PTTG in certain circumstances may explain the discordant expression of PTTG and ID3 in patients 7, 8, and 9 represented in Fig. 4. For example, ID proteins have been shown to be up-regulated in response to TSH (36). Other regulatory stimuli may exist that also modulate the effect of PTTG on ID3 expression in specific circumstances.
ID protein expression has been reported to be a prognostic factor in cervical and breast cancer (28, 37). We correlated ID3 expression with other known clinico-pathological prognostic parameters in thyroid cancer (including TNM staging, tumor type, age at diagnosis, and gender) and observed that although constituting a relatively small cohort (n = 8), follicular thyroid carcinomas were associated with higher ID3 expression than papillary thyroid tumors. We also observed a significantly higher ID3 expression in those over 45 yr old at diagnosis (the threshold age at diagnosis known to be associated with poorer prognosis). Although additional evaluation is necessary with larger cohorts, we propose that measurement of ID3 gene expression has potential for use as a diagnostic and/or prognostic marker in thyroid cancer.
TSP-1 is a multimodular secreted protein that associates with the extracellular matrix and possesses a variety of biological functions, including potent angiogenic activity (38). We have shown TSP-1 expression to be altered in response to manipulation of PTTG expression in vitro. We demonstrated PTTG overexpression to be associated with the down-regulation of TSP-1 in thyroid cells. Conversely, siRNA-targeted inhibition of PTTG expression was associated with the induction of TSP-1 expression. PTTG’s regulation of TSP-1 does not appear to involve the SH3-binding domain, which has been shown to be involved in PTTG’s transactivation of FGF-2, VEGF, and ID3. Although the precise regulatory mechanism involved is not known, we hypothesize that, based on wider evidence, PTTG acts upon TSP-1 expression via intermediary proteins such as ID and p53. p53 is a potent positive regulator of TSP-1 expression (39). PTTG has been shown to inhibit the binding of p53 to DNA and thus suppress its transcriptional activity (40). Therefore, PTTG’s suppression of TSP-1 may involve p53 inhibition in thyroid cells. Recently, ID proteins have been shown to indirectly act upon the TSP-1 promoter and suppress TSP-1 expression. PTTG could also therefore down-regulate TSP-1 via its action upon ID3 expression. Additional studies are necessary to clarify these potential mechanisms of action.
Despite significantly increased expression of PTTG within thyroid cancers, investigation of TSP-1 expression in vivo in our thyroid cancer series demonstrated no significant difference in mean TSP-1 mRNA expression in cancers and normal specimens. Also, analysis of patient-matched normal and thyroid cancer samples demonstrated a highly variable pattern of TSP expression. There exists contradictory evidence for the expression pattern of TSP-1 in thyroid cancer and other tumor types. One study demonstrated TSP-1 expression to be reduced in 50% of papillary, 75% of follicular, and 100% of aggressive undifferentiated thyroid cancers compared with normal tissues (21). However, a more recent report, evaluating 75 papillary thyroid cancers, demonstrated thyroid tumors with high TSP-1 expression to be associated with advanced TNM staging, invasiveness, and degree of angiogenesis (22). The differing results of various studies correlating levels of TSP-1 expression with tumor progression and clinical prognoses are perplexing, and the results from our study emphasize the complex role played by TSP-1 in thyroid cancer. Indeed, the often contradictory and multiple properties of TSP-1 are believed to be due to the interaction of its multifunctional domains and different binding receptors and proteins within the extracellular matrix within the specific tissue site at a given time point, which dictate its predominant local biological effect (38).
Although we did not demonstrate a significant difference in mean TSP-1 expression between tumors and normal specimens, TSP-1 expression showed a significant association with PTTG expression and early tumor recurrence (six tumors recurred within 3 yr of follow-up). Indeed, tumors associated with early recurrence showed 4.2-fold lower TSP-1 expression than normal thyroid tissues. Additional extensive evaluation of TSP-1 as a potential diagnostic or prognostic marker and as a potential therapeutic target in thyroid cancer is thus necessary.
Although extensive studies are required, we provide insight into the angiogenic switch process in thyroid cancer by demonstrating that the protooncogene PTTG may regulate multiple pro- and antiangiogenic genes. The complimentary effects on both of the opposing arms of the angiogenic balance suggest that PTTG may play a major role in thyroid tumor angiogenesis.
|
Footnotes |
|---|
Abbreviations: FGF-2, Fibroblast growth factor-2; ID3, inhibitor of DNA binding-3; PTTG, pituitary tumor-transforming gene; SH3, Src homology domain 3; siRNA, small interfering RNA; TNM, tumor/node/metastases; TSP-1, thrombospondin-1; VEGF, vascular endothelial growth factor; VO-control, empty expression vector alone; WT, wild type.
Received August 12, 2005.
Accepted December 22, 2005.
|
References |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
B survival pathway signaling. Blood 104:1465–1473
inhibits expression of minichromosome maintenance proteins in vascular smooth muscle cells. Mol Endocrinol 17:1005–1018This article has been cited by other articles:
 |
|
 |
|
  M. A. Morris, C. W. Dawson, W. Wei, J. D. O'Neil, S. E. Stewart, J. Jia, A. I. Bell, L. S. Young, and J. R. Arrand Epstein-Barr virus-encoded LMP1 induces a hyperproliferative and inflammatory gene expression programme in cultured keratinocytes J. Gen. Virol., November 1, 2008; 89(11): 2806 - 2820. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Salehi, K. Kovacs, B. W Scheithauer, R. V Lloyd, and M. Cusimano Pituitary tumor-transforming gene in endocrine and other neoplasms: a review and update Endocr. Relat. Cancer, September 1, 2008; 15(3): 721 - 743. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ito, Y. Shimada, T. Kan, S. David, Y. Cheng, Y. Mori, R. Agarwal, B. Paun, Z. Jin, A. Olaru, et al. Pituitary Tumor-Transforming 1 Increases Cell Motility and Promotes Lymph Node Metastasis in Esophageal Squamous Cell Carcinoma Cancer Res., May 1, 2008; 68(9): 3214 - 3224. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M El-Naggar, M. T Malik, A. Martin, J. P Moore, M. Proctor, T. Hamid, and S. S Kakar Development of cystic glandular hyperplasia of the endometrium in Mullerian inhibitory substance type II receptor-pituitary tumor transforming gene transgenic mice J. Endocrinol., July 1, 2007; 194(1): 179 - 191. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. S. Kim, H. Ying, M. C. Willingham, and S.-y. Cheng The pituitary tumor-transforming gene promotes angiogenesis in a mouse model of follicular thyroid cancer Carcinogenesis, May 1, 2007; 28(5): 932 - 939. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Vlotides, T. Eigler, and S. Melmed Pituitary Tumor-Transforming Gene: Physiology and Implications for Tumorigenesis Endocr. Rev., April 1, 2007; 28(2): 165 - 186. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. S. Kim, J. A. Franklyn, K. Boelaert, M. C. Eggo, J. C. Watkinson, and C. J. McCabe Pituitary Tumor Transforming Gene (PTTG) Stimulates Thyroid Cell Proliferation via a Vascular Endothelial Growth Factor/Kinase Insert Domain Receptor/Inhibitor of DNA Binding-3 Autocrine Pathway J. Clin. Endocrinol. Metab., November 1, 2006; 91(11): 4603 - 4611. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
