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Departments of Surgery (M.C.S., O.N.T., M.H., D.G., L.T., T.J.F.), New York Presbyterian Hospital and Weill Medical College of Cornell University, New York, New York 10021; and Strang Cancer Prevention Center (T.J.F.), New York, New York 10021
Address all correspondence and requests for reprints to: Thomas J. Fahey III, M.D., New York Presbyterian Hospital-Cornell University Room F-2024, 525 East 68 Street, New York, New York 10021. E-mail: tjfahey{at}mail.med.cornell.edu
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Abstract |
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Introduction |
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Recent studies have established the presence of two distinct COX enzymes, a constitutive enzyme (COX-1) and an inducible form (COX-2). Cyclooxygenases catalyze the formation of prostaglandins from arachidonic acid. COX-1 is thought to be a housekeeping gene with essentially constant levels of expression, whereas COX-2 is an early-response gene that, like c-jun and c-fos, is induced rapidly by growth factors, tumor promoters, oncogenes, and carcinogens (5).
Multiple lines of evidence suggest that COX-2 is important in carcinogenesis. COX-2 is up-regulated in transformed cells (6) and in many epithelial carcinomas including; colon, stomach, pancreas, and prostate (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18), whereas levels of COX-1 are relatively constant. Moreover, a null mutation for COX-2 caused a marked reduction in the number and size of intestinal polyps in APC
716 mice, a murine model of familial adenomatous polyposis (19). COX-2 knockout mice also developed about 75% fewer chemically induced skin papillomas than control mice (20). In addition to the genetic evidence implicating COX-2 in carcinogenesis, newly developed selective inhibitors of COX-2 protect against gastrointestinal tumor formation (19, 21, 22).
In this study, we investigated whether COX-2 expression was increased in thyroid cancers. Our data reveal that levels of COX-2 mRNA and protein are increased in human thyroid cancer compared with adjacent normal tissue. In addition, we demonstrate that COX-2 mRNA expression can be detected in fine needle aspiration (FNA) specimens and therefore suggest that COX-2 may serve as a marker of carcinoma.
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Materials and Methods |
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Roswell Park Memorial Institute medium 1640, FBS, penicillin, streptomycin, COX-2, and ß2-microglobulin primers were from Life Technologies, Inc. (now Invitrogen, Grand Island, NY). RNeasy Mini kits were from QIAGEN Inc. (Santa Clarita, CA). Lowry protein assay kits, phorbol 12-myristate 13-acetate (PMA), and secondary antibody to IgG conjugated to horseradish peroxidase were from Sigma (St. Louis, MO). The COX-2 standard for immunoblotting was from Cayman Chemical Co. (Ann Arbor, MI). The COX-2 polyclonal antibody, PG-27, was from Oxford Biomedical Research, Inc. (Oxford, MI). Nitrocellulose membranes were from Schleicher \|[amp ]\| Schuell, Inc. (Keene, NH). Western blotting detection reagents (electrochemiluminescence) were from Amersham Pharmacia Biotech (Buckinghamshire, UK).
Patient samples
Biopsy specimens were obtained at the time of surgery from 28 patients with thyroid nodules. Specimens were obtained chronologically over a 3-month period. Tissue samples were taken from a nonnecrotic area of the tumor and from adjacent grossly normal tissue; samples were immediately frozen in liquid nitrogen, and subsequently stored at -80 C. The benign thyroid nodules included 8 follicular adenomas, 1 Hurthle cell adenoma, 2 nodular goiters, and 2 patients with hyperplasia. The malignant nodules included 12 papillary carcinomas, 1 medullary carcinoma, and 1 follicular carcinoma.
FNA specimens were obtained at the time of surgery using a 25-gauge needle. After aspiration, the needle was flushed with approximately 300 µl of sterile saline and placed on ice. Cell specimens were centrifuged and the pellet stored at -80 C until time of assay. This study was approved by the Committee on Human Rights in Research at Weill Medical College of Cornell University.
Tissue culture
Three human thyroid cancer cell lines FRO (follicular neoplasm), ARO (anaplastic neoplasm), and NPA (papillary neoplasm) were established by Dr. Juillard (UCLA). The three cell lines were maintained in Roswell Park Memorial Institute medium 1640 supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were plated for experimental use in complete media and allowed to attach and grow for 48 h in a 5% CO2/water saturated incubator at 37 C. After washing with PBS, the media was then replaced with serum-free media. Twenty-four hours later, cells were treated with vehicle or PMA under serum-free conditions.
RNA isolation and reverse transcription
Total RNA was isolated from thyroid tissue (
50 mg), FNA specimens, and cell monolayers using RNeasy Mini Kits from QIAGEN. One microgram of total RNA was reverse transcribed using the GeneAmp RNA PCR kit according to the manufacturer’s protocol. The entire amount of total RNA extracted from FNA specimens was reversed transcribed.
Construction of a COX-2 competitor template containing a nucleotide deletion
A competitive RT-PCR deletion construct (mimic) for COX-2 was synthesized using a mutant sense primer ]nucleotides (nt) 932–955 attached to nt 1111–1130; 5'-GGTCTGGTGCCTGGTCTGA TGATGGAGTGGCTATC ACTTCAAAC-3'] and an antisense primer (nt 1634–1655; 5'-GTCCTTTCAAGGAGAATGGTGC-3'), producing a 569-bp PCR product (12). The mutant sense primer contains the primer-binding sequence of endogenous target (from nt 932–955) attached to the end of an intervening DNA sequence (a 156-bp deletion from nt 956-1110). Thus, the mimic DNA has primer binding sequences identical to the target cDNA. The 569-bp mimic was further amplified using the sense primer (5'-GGTCTGGTGCCTGGTCTGATGATG-3') and the antisense primer (5'-GTCCTTTCAAGGAGAA TGGTGC-3') in a reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM deoxynucleotide triphosphate, 2.5 U AmpiTaq DNA polymerase and 400 nM primers for 35 cycles consisting of denaturation at 94 C for 20 sec, annealing at 60 C for 20 sec, and extension at 72 C for 30 sec in a Perkin-Elmer Corp. 2400 thermal cycler. The PCR products were electrophoresed on 1% agarose gels and gel-purified using GenElute Agarose Spin Columns (Perkin Elmer, Branchburg, NJ) according to the manufacturer’s protocol.
Quantitative PCR for COX-2 in human thyroid tissue
Each PCR was carried out in 25 µl of reaction mix, containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM deoxynucleotide triphosphate, 2.5 U Amplitaq DNA polymerase (Perkin Elmer) and 400 nM primers (sense: 5'-GGTCTGGTGCCTGGTCTGATGATG-3' and antisense: 5'-GTCCTTTCAAGGAGAATGGTGC-3'). Five microliters of reverse-transcribed cDNA samples and various known amounts of COX-2 mimic (between 0.0001 and 0.05 pg) adjusted to the abundance of the target cDNA, were added to the reaction mix and coamplified for 35 cycles: denaturation at 94 C for 20 sec, annealing at 65 C for 20 sec, extension at 72 C for 90 sec, and final extension at 72 C for 10 min. Ten microliters of PCR products, 724-bp fragments from endogenous target cDNA and 569-bp fragments from mimic COX-2, were then separated by electrophoresis on 1% agarose gels and visualized by ethidium bromide staining.
Densitometry was performed using Eagle Eye software (Stratagene, La Jolla, CA). A standard curve was generated and COX-2 expression levels calculated using Microsoft Corp. (Redmond, WA) Excel.
Semiquantitative PCR for COX-2 and ß2-microglobulin in thyroid cell lines
The semiquantitative analysis for COX-2 was performed using the same COX-2 primers as listed above in a 25-µl reaction mixture containing 5-µl aliquots of reverse transcribed cDNA samples, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM deoxynucleotide triphosphate, 2.5 U AmpliTaq DNA polymerase, and 400 nM primers for 35 cycles consisting of denaturation at 94 C for 20 sec, annealing at 65 C for 20 sec; extension at 72 C for 30 sec and final extension at 72 C for 10 min. A constitutively expressed gene, ß2-microglobulin was used as an internal control, generating a 266-bp PCR product. The primers for ß2-microglobulin (from nt 75–340) were 5'-AGCAGAGAATGGAAAGTCAAA-3' sense and 5'-ATGCTGCTTACATGTCTCGAT-3' antisense. The PCR conditions for ß2-microglobulin were identical to that for COX-2 except for annealing at 55 C for 20 sec.
Western blotting
Frozen tissue was thawed in ice-cold homogenization buffer containing 150 mM NaCl, 100 mM Tris-buffered saline (pH 8), 1% Tween-20, 50 mM diethyldithiocarbamate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, aprotinin, trypsin-chymotrypsin inhibitor, and pepstatin. Tissues were homogenized using a glass-on-glass tissue homogenizer. Homogenates were centrifuged at 11,750 x g for 10 min at 4 C to remove the particulate material.
Cellular lysates were prepared by treating cells with the same lysis buffer that was used for the tissue samples. Lysates were incubated for 20 min. on ice and centrifuged at 11,750 x g for 10 min to sediment the particulate material. The protein concentration of the supernatant was measured using the Lowry protein assay kit (Sigma). Immunoblot analysis for COX-2 was performed. One hundred micrograms/lane of protein from tissue and 30 µg/lane of protein from cellular lysates were loaded on a 10% SDS-PAGE gel, after transfer, the membrane was blocked in 3% BSA and incubated over night with 1:1000 COX-2 antibody. ß-actin was used as an internal control.
Immunohistochemistry and immunofluorescence
Tissues from six patients with thyroid nodules were fixed in formalin, embedded in paraffin, cut into 4-µm sections and mounted onto polylysine coated slides. Sections were dewaxed in xylene, rehydrated in descending alcohols, and blocked for endogenous peroxidase (3% H2O2 in methanol) and avidin/biotin (Vector Blocking Kit, Vector Laboratories, Inc., Burlingame, CA). The sections were permeabilized in 0.1 M Tris pH 7.5, 0.15 M NaCl, 0.5% blocking agent, 0.3% Triton-X, 0.2% saponin (TNB-BB), blocked in 3% BSA, and incubated in primary antibody overnight at 4 C. The polyclonal antiserum to COX-2 (PG-27, Oxford Biomedical Research, Inc.) was used at a 1:500 dilution in TNB-BB. Control sections were incubated with antisera in the presence of a 100-fold excess of human recombinant COX-2 protein, or with isotype matched IgG normal rabbit serum. Immunoreactive complexes were detected using tyramide signal and amplification-indirect and visualized with the peroxidase substrate, diaminobenzidine. Slides were then counter stained in aqueous hematoxylin, mounted in crystal mount, and coverslipped in 50:50 xylene/Permount (Fisher Scientific, Pittsburgh, PA). Thyroid cell lines grown on chamber slides were prepared in a similar fashion and then incubated with fluorescein isothiocyanate-conjugated second antibodies from Sigma (goat antirabbit IgG, diluted 1:80) for 1 h, followed by washing with PBS. Slides were then counter stained with propidium iodide and viewed with fluorescence microscopy.
Statistical analysis
Results were analyzed by the Wilcoxon Signed Rank test. A difference P less than 0.05 between groups was considered significant.
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Results |
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To analyze the expression of COX-2, we used a competitive RT-PCR assay in which the amount of COX-2 mRNA could be measured from minute quantities of total RNA (0.5 µg RNA). The competitor (569-bp) and target (724-bp) use the same PCR primers but yield amplicons with a different size (Fig. 1A
), allowing their separation on a gel at the end of the reaction. There was no statistical significance in the benign nodules (mean 12.6 ± 22 fg/mg total RNA) vs. its adjacent normal tissue (mean, 9.5 ± 14.5 fg/mg total RNA) tissue; P = 0.66. Similarly, levels of COX-2 mRNA in 14 pairs of malignant nodules and adjacent thyroid tissue were compared. Amounts of COX-2 mRNA in malignant nodules (mean 91.2 ± 110.6 fg/mg total RNA) vs. its adjacent normal tissue (mean 10.8 ± 9.5 fg/mg total RNA) demonstrated statistical significance; P = 0.017. There was no notable difference in COX-2 expression among the different types of malignant thyroid tumors due to the small sample number. In addition, COX-2 mRNA levels were significantly increased in malignant thyroid nodules compared with benign thyroid nodules; P = 0.01 (Fig. 1B)
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) All 8 of these patients had increased COX-2 mRNA expression by quantitative RT-PCR.
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). In tumor tissue, expression of COX-2 was localized to tumor cells, but not to surrounding stromal cells or infiltrated inflammatory cells (Fig. 3B). The staining pattern was predominantly granular and in the perinuclear cytoplasmic region. This staining was specific for COX-2 as immunoreactivity was lost when the antiserum to COX-2 was preabsorbed with human recombinant COX-2. Immunofluorescent analysis of the three thyroid cancer cell lines, demonstrated diffuse, cytoplasmic predominantly perinuclear staining (Fig. 3C).
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To determine if the up-regulation of COX-2 mRNA and protein seen in thyroid tumors was reproducible in vitro, we analyzed three human thyroid cancer cell lines, FRO, NPA, and ARO. As shown in Fig. 4, COX-2 mRNA and protein were detected under basal conditions in the ARO cell line. Treatment with PMA, a tumor promoter, induced COX-2 mRNA and protein in as early as 3 h. Similar results were obtained in the FRO and NPA cell lines (data not shown).
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To determine if mRNA transcripts of COX-2 could be detected in FNA specimens by RT-PCR, we analyzed RNA from a FNA specimen and serial dilutions of cells from a thyroid cancer cell line. Figure 5 demonstrates mRNA COX-2 expression in serial dilutions of a thyroid cancer cell line (NPA). COX-2 mRNA was detectable in as few as 50 thyroid cancer cells. Additionally, both an FNA specimen and the cells remaining in the needle after cells for cytology were removed clearly demonstrate mRNA COX-2 expression.
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Discussion |
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The cyclooxygenase pathway has been implicated as a mediator of inflammation and cellular growth in the thyroid. Early studies suggest that in FRTL-5 cells cyclooxygenase activity is increased with growth stimulation (23). The inducible form of cyclooxygenase, COX-2 and its role in inflammation of the thyroid was first described by Di Paola et al. (24). They demonstrated that IgG-mediated cellular proliferation of FRTL5 thyroid cells was attenuated with a nonspecific cyclooxygenase inhibitor, indomethacin. Similarly, IL-1ß and TNF
, two well known proinflammatory mediators of thyroiditis, induced COX-2 expression in thyroid epithelial cells (25). Finally, Smith, et al. ( 26) sought to evaluate expression of COX-2 in thyroid tissue. They demonstrated constitutive expression of COX-2 by immunohistochemistry, immunoblot, and Northern analysis in tissue from a multinodular goiter, Graves’ disease and a papillary carcinoma. These studies, however, did not examine whether COX-2 levels are differentially expressed in benign vs. malignant tissues. Using quantitative PCR techniques, we were able to detect a significant difference in the level of COX-2 mRNA and protein in malignant thyroid tissue compared with both adjacent normal thyroid tissue and benign thyroid nodules. Moreover, benign thyroid nodules had COX-2 expression comparable to its adjacent normal thyroid tissue.
Six of the papillary carcinomas had COX-2 mRNA expression above 100 fg/mg total RNA. An absolute value above this number may be proposed to correlate with a malignant tumor. However, a much larger study with increased sample size is necessary to confirm this number.
Activation of cyclooxygenase-2 is theorized to promote carcinogenesis via multiple mechanisms. Enhanced synthesis of prostaglandins, a consequence of up-regulation of COX-2, favors the growth of malignant cells by increasing cellular proliferation (27), promoting angiogenesis (28) and inhibiting immune surveillance (29). In intestinal epithelial cells, overexpression of COX-2 inhibits apoptosis (30) and increases the invasiveness of malignant cells (31).
There are many known genetic alterations associated with thyroid cancer, which could account for an increase in COX-2 expression. The prevalence of a ras oncogene mutations in thyroid carcinomas has been reported to be as high as 92%. Ras mutations have been found in both benign and malignant epithelial-derived thyroid tumors, including follicular adenomas, follicular carcinomas, papillary thyroid carcinomas and anaplastic carcinomas (32, 33). Fibroblasts transformed with a mutant Ha-Ras oncogene responds with a rapid induction of COX-2 (34). Similarly, Ha-ras expression in intestinal epithelial cells lead to expression of COX-2 (35). Therefore, the high prevalence of ras mutations in thyroid tumors may lead to downstream activation and overexpression of COX-2.
The RET protooncogene also contributes to the pathogenesis of papillary carcinomas, sporadic medullary carcinomas, and familial medullary thyroid carcinomas (2). It has yet to be demonstrated whether activation of RET by the RET/PTC translocations leads to induction of COX-2. However, RET activation has been previously shown to activate ras and thus could indirectly lead to COX-2 activation (36). Future studies may elucidate a link between RET activation and COX-2 up-regulation, either directly or indirectly through the ras pathway.
Although APC mutations are uncommon in sporadic thyroid tumors, there is an increased incidence of thyroid tumors in familial adenomatous polyposis FAP and Gardner’s syndrome (37). Researchers recently demonstrated a link between COX-2 and APC in mice carrying a mutation of the APC gene. These mice were protected against tumor formation in the setting of COX-2 deficiency (19). Therefore, APC mutations may represent a rare mechanism for COX-2 up-regulation in familial adenomatous polyposis-associated thyroid tumors.
Fine needle aspiration has become a critical component in the management of thyroid nodules. The use of fine needle aspiration has decreased the number of thyroid nodules requiring surgical treatment from 67% to 44%. Similarly, the percentage of carcinomas in those nodules that are operated on has increased from 14% to 29% (38). Preoperative discrimination of benign and malignant thyroid nodules would be useful either for eliminating the need for surgery for some thyroid nodules or for guiding the extent of surgery for other thyroid nodules. There has been recent interest in using molecular markers to enhance the cytopathologic diagnosis of thyroid nodules by FNA (39). To date, however, there is no single marker identified that has been shown to consistently predict benign or malignant final pathology. Ultimately, it is likely that a combination of molecular markers will be useful as an adjunct to cytopathology in the diagnosis and treatment of thyroid nodules. Overexpression of COX-2 in thyroid nodules and the ability to detect COX-2 by FNA suggest that COX-2 analysis of thyroid nodule FNA may be a useful adjunct to cytopathology.
Recent studies with celecoxib, a selective COX-2 inhibitor, indicate that blocking COX-2 inhibits neoangiogenesis and tumor growth of lung and colon tumors in mice (40). This report and others suggest that COX-2 expression is an important contributor to the pathogenesis of some, if not all, epithelial cancers. Based on our findings of increased expression of COX-2 in malignant thyroid tumors, it will be important to establish whether COX-2 inhibitors may play a role in the prevention or treatment of thyroid malignancy.
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Acknowledgments |
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Footnotes |
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Abbreviations: ARO, Anaplastic neoplasm; COX-1, constitutive cyclooxygenase; COX-2, inducible cyclooxygenase; FNA, fine needle aspiration; FRO, follicular neoplasm; NPA, papillary neoplasm; nt, nucleotide(s); PMA, phorbol 12-myristate 13-acetate.
Received June 14, 2001.
Accepted October 9, 2001.
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716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell 87:803–809[CrossRef][Medline]
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