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The Ohio State University and Arthur G. James Comprehensive Cancer Center (N.S., A.V.E., M.S., V.V.V., M.D.R.), Columbus, Ohio 43210; Washington Hospital Center/MedStar Research Institute (N.S., V.V.V., K.D.B., M.D.R.), Washington, D.C. 20010; Uniformed Services University of the Health Sciences (V.V.V., M.D.R.), Bethesda, Maryland 20814; and Section on Endocrinology and Genetics (I.B., C.A.S.), DEB, National Institute of Child Health and Development, National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Matthew D. Ringel, M.D., The Ohio State University, 445D McCampbell Hall, 1581 Dodd Drive, Columbus, Ohio 43210. E-mail: ringel.11{at}osu.edu.
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Abstract |
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Design and Results: We used GPR54-null thyroid cancer cells to create a stable GPR54 overexpression model. Cell growth and cell migration of the GPR54-expressing lines were inhibited by recombinant metastin, and metastin stimulated the protein kinase C, ERK, and phosphatidylinositol-3-kinase pathways. To identify metastin-regulated genes, we performed microarray analyses using RNA isolated from GPR54 stable transfectants before and after 1 and 24 h of metastin stimulation. Consistent increases in expression of the gene encoding myocyte-enriched calcineurin interacting protein 1 (MCIP-1), an inhibitor of calcineurin, were identified and confirmed using real-time RT-PCR and Western blot. Functionally, metastin treatment of GPR54-expressing cells initially increased calcineurin activity, followed by a prolonged reduction in calcineurin activity for 24 and 48 h, consistent with the pattern of MCIP-1 expression. In addition, treatment with cyclosporin A, a calcineurin inhibitor, blocked cell migration. Lymph node metastasis in papillary thyroid cancers demonstrated loss of MCIP-1 expression in comparison with primary tumors.
Conclusions: These data suggest a role for MCIP-1 and calcineurin inhibition in GPR54-mediated metastasis suppression in human cancers.
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Introduction |
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The KiSS-1gene (U43527) located on chromosome 1 encodes a metastasis suppressor that was isolated from melanoma cells (2). The proteins encoded by this gene, termed kisspeptins or metastin (3, 4, 5), have been shown to have antimigratory effects in vitro and metastasis-inhibiting effects in vivo (2). The KiSS-1gene products have been identified as the endogenous ligands for a heptahelical G protein-coupled receptor (GPR54) (3, 4, 5). This receptor couples primarily to G
q/11, leading to release of intracellular calcium, activation of protein kinase C (PKC), and p38 MAPK. The expression of GPR54 mRNA has been identified in many human tissues, including placenta and the central nervous system. Recently, mutations in the human GPR54 gene were found to be responsible for hypogonadotropic hypogonadism (6). Animal studies confirmed that expression of GPR54 is essential for murine pubertal development (7).
GPR54 expression has been reported in esophageal (8), pancreatic (9), bladder (10), melanoma (11), hepatocellular (12), and thyroid (13) cancer tissue. We reported that GPR54 mRNA expression was maintained in primary papillary thyroid cancers and was reduced in follicular thyroid cancers, consistent with the greater tendency of follicular thyroid cancer to metastasize hematogenously (13); however, the expression levels or pattern of GPR54 protein were not assessed. Activation of endogenous GPR54 in ARO thyroid cancer cells with metastin resulted in increased PKC activity as well as enhanced p42/44 MAPK and Akt (PKB) activation (13).
Activation of PKC, intracellular calcium release, MAPK, and Akt have all been associated with thyroid tumorigenesis and/or progression (reviewed in Refs. 14 and 15). For this reason, the precise mechanism for GPR54-mediated metastasis inhibition in thyroid cancer cells has been uncertain. To better characterize the mechanism of action of metasin/GPR54 in thyroid cancer cells, we created a GPR54 overexpression model in GPR54-null thyroid cancer cells to identify GPR54-regulated genes that are potentially responsible for its antimetastasis effect using cDNA microarray. We determined that GPR54 activation enhanced the expression of myocyte-enriched calcineurin interacting protein 1 (MCIP-1), a well-characterized inhibitor of calcineurin (reviewed in Ref. 16). Additional studies demonstrated that metastin reduced calcineurin activity in GPR54-overexpressing thyroid cancer cells and that metastatic thyroid cancer tissue is characterized by loss of MCIP-1 expression, consistent with escape from a metastasis inhibitory effect.
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Materials and Methods |
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Recombinant metastin, cDNA encoding GPR54, and a polyclonal antimetastin antibody were gifts from Takeda Chemical Industries, Ltd. (Osaka, Japan). cDNA encoding MCIP-1 in pTarget expression vector was the generous gift of Dr. Beverly Rothermel (University of Texas, Dallas, TX). PCR-related material was purchased from Applied Biosystems (Foster City, CA). Random hexamers, primers, and sequence-specific probes for PCR were from QIAGEN, Inc. (Valencia, CA). Buffers for PCR, TRIzol, culture medium, and serum were obtained from Invitrogen (Carlsbad, CA). Primary antibodies against GPR54 were from Takeda and from Phoenix Pharmaceuticals (Belmont, CA). Primary antibodies for MCIP-1 (AOO34 and A1198) were obtained from The University of Texas Southwestern Program for Genomic Applications (Dallas, TX). Primary antibodies recognizing phosphorylated PKC, phosphorylated and total Akt, and phosphorylated and total p42/44 MAPK as well as U0126 and LY294002 were from Cell Signaling (Beverly, MA). -Tubulin antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Cyclosporine was from EMB Biosciences, Inc. (San Diego, CA). All other materials were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise described.
Human tissue samples
Human thyroid cancer samples were obtained using an Institutional Review Board-approved protocol from The Hospital for Endocrine Surgery, Kiev, Ukraine, after informed consent was obtained. The use of these samples was also approved by the Institutional Review Board at Uniformed Services University of the Health Sciences, where these samples were analyzed. All tumors were grossly invasive and had evidence of nodal metastasis.
Cancer cell lines and stable transfection
Human thyroid carcinoma ARO (anaplastic) and NPA (papillary) cell lines were obtained from Dr. Guy J. F. Juillard (University of California, Los Angeles, Los Angeles, CA) and were grown in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum (FCS). We have previously demonstrated that ARO cells express endogenous GPR54, whereas NPA cells do not (13). NPA thyroid cancer cells were transfected with a GPR54 cDNA insert in pcDNA3.1(+) (Invitrogen). After selection of transfected clones by G418 resistance, clones were screened for expression of GPR54 by RT-PCR. Twenty-three expressing clones were identified, as were two that expressed vector alone. Experiments were subsequently performed using nontransfected NPA cells, vector-expressing NPA cells, and three clones with low, medium, and high levels of GPR54 mRNA compared with ARO cell (endogenous) levels.
Cell culture
Cells were grown, plated in 10-cm dishes, and grown in RPMI 1640 medium containing 10% FCS. Before metastin-stimulation experiments, the growth medium was aspirated and replaced with serum-free RPMI 1640 for 24 h, and this medium was aspirated and replaced with 5 ml fresh serum-free RPMI 1640 for 4 h at which time metastin was added to the medium. For blocker experiments, the calcineurin inhibitor was added 30 min before the metastin.
RNA isolation
After medium aspiration, cells were washed with ice-cold PBS twice, and 1 ml TRIZOL (Invitrogen) was added. Total RNA isolation was performed as per the manufacturer’s recommendation. For cDNA microarray analysis, RNA was further cleaned by using RNeasy maxi kit (QIAGEN).
Real-time RT-PCR
Real-time RT-PCR was performed for several transcripts, including GPR54, to determine the expression levels in the cell model as described previously in detail (13). MCIP-1 and TRB-3 (primers and sequence-specific probes from Applied Biosystems) were amplified using the same methods. As a control for RNA integrity and for assay normalization, 18S rRNA was amplified using TaqMan rRNA control reagents kit as described (13).
GPR54, MCIP-1, TRB3, and 18S were amplified from all samples in duplicate in two separate reactions. Negative controls are included for the RT-PCR and for the PCR alone in each reaction. To quantify results, standard curves were created using serial dilutions of RNA isolated from either ARO cells for GPR54 or HEK 293 transiently transfected with MCIP-1 cDNA. 18S was also normalized using these same standards. Normalized levels were calculated as the ratio of target mRNA to 18S. Several samples were run on electrophoreses through agarose gels, and all showed a single unique band at the expected sizes.
Immunohistochemistry
Cells were cultured in LabTek chamber slides (Nalge Nunc International, Naperville, IL). At low confluence levels (
10–15%), the RPMI 10% FCS medium was aspirated and cells were fixed using 3% formalin solution. After washing in PBS, cells were incubated with 0.2% Triton solution. Incubation with GPR54 or MCIP-1 antibody was performed overnight at 4 C. Secondary antibody was applied for 1 h in the dark at room temperature. Cover slides were then mounted with Prolong antifade kit (Molecular Probes, Eugene, OR) and subjected to microscopic examination.
Protein isolation and Western blotting
Protein lysates were isolated as previously described in detail (13, 17). Twenty micrograms of total lysate were subjected to 8% or 4–10% SDS-PAGE, and the separated proteins were transferred to nitrocellulose membranes by electrophoretic blotting. Nonspecific binding was prevented using Tris-buffered saline with Tween 20 containing 5% nonfat dry milk overnight at 4 C. Immunoblotting was performed as previously described (13, 17). Relative quantitation of proteins was determined by scanning and band densitometry using ImageGauge software (Fuji Photo Film Co., Tokyo, Japan). Immunoblots of the same protein samples were performed for -tubulin to verify equal loading and for normalization for quantitation between lanes.
Cell proliferation
Cells were plated in 12-well plates in growth medium containing serum and were cultured in RPMI 1640 without serum for 1 d. Cells were then incubated with or without various doses of metastin and/or various concentrations of pharmacological inhibitors of intracellular pathways. After 1–3 d, DNA in each plate was precipitated with perchloric acid and incubated with diphenylamine reagent, and DNA content was measured by spectrophotometry to assess cell proliferation as previously described in detail (18).
Migration experiments
Cell migration experiments were performed and quantified as previously described in detail (17). Briefly, 300 µl of cell suspension (final concentration of 3 x 105 per well) was placed on the top of the membrane of a Boyden chamber (8-µm pore size) in serum-free RPMI medium. The bottom part of the chamber was filled with 400 µl serum-free RPMI 1640. After 1 h of incubation, medium was aspirated from both chambers. Serum-free RPMI 1640 medium (300 µl) containing metastin and/or pharmacological inhibitors was added to the top chamber and RPMI 1640 containing 5% fetal bovine serum (400 µl). Cells were then incubated for 16 h. At the end of the experiments, cells were fixed and stained and were observed using both low-power (x10) and high-power (x40) microscope objectives. Photographs of four representative high-power fields of the total membrane-bound cells were taken. Nonmigrated cells remaining on the top of the membranes were removed using a cotton swab. Cells that were not removed are those that migrated to the lower membrane. Digital photographs were obtained and transformed into gray-scale and then to a bit map using 50% threshold imaging (Adobe Photoshop 7). The percentages of cells migrated were then calculated using the mean and SD of the four photographs for the total and migrated cells on each membrane. All migration experiments were performed on at least three occasions in duplicate.
Calcineurin activity
Cellular calcineurin activity was measured using a colorimetric assay from EMD Biosciences, Inc. (San Diego, CA) using RII phosphopeptide substrate as per the manufacturer’s recommended protocol. Cells were grown in 10-cm dishes in RPMI medium containing 10% FCS until subconfluent, serum depleted for 24 h, and then stimulated with 1 µM metastin or vehicle control. To terminate the metastin stimulation, cells were washed with Tris-buffered saline in phosphate-free conditions. After isolating protein and removing potential phosphate contamination as per the manufacturer’s recommended method, phosphatase activity was measured as total phosphatase activity, phosphatase activity in the presence of okadaic acid [OA, which inhibits phosphatase activity but has no effect on calcineurin and protein phosphatase (PP)2C], and phosphatase activity in the presence of okadaic acid and EGTA (OA+EGTA, which inhibits all phosphatase activity except PP2C). Calcineurin activity (CaN) is then calculated using the formula: CaN (PP2B) = OA – (OA+EGTA).
NFAT (nuclear factor activated in T-lymphocytes) transcription assay
Cells were seeded on six-well plates and incubated in RPMI 1640 with 10% FCS until 50% confluent. Transfection of NFAT-luciferase (gift from Dr. Michael Levine, Cleveland Clinic Foundation, Cleveland, OH) and pRL-SV (Renilla luciferase) were performed as per the manufacturer’s recommended protocol (Promega, Madison, WI). Cells were transfected with the reporter constructs for 4 h, and 48 h after transfection, and after washing cells at 24 and 48 h, metastin (1 µM) was added to the medium. After 6 h, cells were washed, incubated with the Passive lysis buffer from the dual luciferase reporter assay system kit (Promega) at room temperature, and lysates were collected. Twenty microliters of each sample and 100 µl of the luciferase assay substrate reagent II (dual luciferase reporter assay system kit; Promega) were added into a 96-well plate, and firefly luminescence was measured by luminometer followed by adding 100 µl of the Stop and Glo reagent (Promega) to measure Renilla luciferase activity.
Statistical analysis
The effects of metastin on DNA synthesis, migration, luciferase activity, and calcineurin activity were examined by Student’s t test or by nonparametric tests depending on the data distribution using StatView (Abacus Concepts Inc., Berkeley, CA). Experiments were repeated at least three times in duplicate. For all analyses, P < 0.05 was considered significant.
Sample labeling and hybridization for cDNA microarray
Briefly, total RNA (20 µg) from GPR54-expressing NPA cancer cells was isolated before and 1 and 24 h after stimulation with recombinant metastin (1 µM) and was used for microarray analysis. Labeling was performed by RT reaction with green fluorescent cyanine 3-dUTP (Cye3) and red fluorescent cyanine 5-dUTP (Cye5) using the MICROMAX direct cDNA microarray system (NEN Life Science Products Life Sciences Inc., Boston, MA) as previously described (19). Samples were combined, purified, and concentrated with Microcon YM30 columns (Millipore Corp., Bedford, MA). After denaturation, probes were hybridized at 65 C for 16 h to microarray glass slides that were made at the National Cancer Institute (Bethesda, MD) core facility containing approximately 12,000 human PCR-amplified cDNA clones. Slides were then washed and scanned on a dual-laser microarray scanner (GenePix 4000; Axon Instruments, Foster City, CA). The microarrays were performed in duplicate, including reciprocal labeling of fluorochromes. These images were analyzed by GenePix Pro3.0 (Axon Instruments). Data were imported in the National Cancer Institute Center for Information Technology Microarray database (NCI-CIT µArray).
Microarray data analysis
Data analysis was performed as described previously (19). After excluding all spots with defective hybridization, Cye3 and Cye5 fluorescent intensities and background values were obtained for each individual spot using the GenePix 4000 microarray software. For each spot, the signal intensities were determined by the following formula: (mean spot intensities) – (median background intensities).
Only spots with signal intensities of at least 500 pixels (after background subtraction) in at least one channel were included. We empirically determined the significance cutoff for signal to background ratio as 1.5 in both channels. For each spot, the average replicates for ratios were calculated for individual samples. Each slide was normalized based on the information available at the NCI-CIT µArray site (http://nciarray.nci.nih.gov). Differences in gene expression between the basal and metastin-stimulated samples were determined. Genes were considered to be significantly up- or down-regulated if the fold differences between the expression before and after metastin stimulation was more than 2.0 in all replicates.
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Results |
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Preliminary studies were performed to determine the expression levels of endogenous GPR54 in three thyroid cancer cell lines. Of these three lines, ARO expresses endogenous receptor and the NPA cell line had no detectable endogenous GPR54 mRNA (Fig. 1
). Thus, we used the NPA line for the stable expression model. After transfection and selection, expression of GPR54 was examined using real-time quantitative RT-PCR. Three clones (clones 9, 19, and 23) were isolated, and data from one representative positive clone with intermediate mRNA expression levels (clone 19) are shown in Fig. 1. Experiments were performed with all three cell lines that expressed different levels of GPR54 mRNA by quantitative RT-PCR. Clone 13 expressed vector alone, with no detectable GPR54, and served as a vector control. We further confirmed the expression of GPR54 protein in these cells by immunofluorescence staining for GPR54 (Fig. 1). Similar levels of immunoactive GPR54 were detected for the three stable transfectant cell lines (data not shown). Antibodies raised against GPR54 were not suitable for Western blot detection (data not shown). All presented data were obtained using three GPR54-expressing cell lines and vector-expressing control cells.
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To confirm that the transfected GPR54 was functioning, GPR54-expressing cells were incubated with 1 µM metastin for 24 h for a time-course experiment. Initial dose-response studies in transfected cells and in ARO cells that express endogenous GPR54 were performed. Activation of pathways was demonstrable beginning with concentrations of 500 nM metastin, with maximal effect noted using 1 µM (13). Similar to the ARO cells, metastin incubation resulted in phosphorylation of Akt, p42/44 MAPK, and PKC (Fig. 2). No significant changes in total protein levels of Akt, p42/44 MAPK, or PKC were identified. Stimulation of the null transfectants or the wild-type NPA resulted in the activation of none of these pathways.
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To assess the biological function of the transfected GRP54 in NPA thyroid cancer cells, we measured DNA content of wells as a measure of cell number and assessed cell migration using Boyden chamber assays. Cells were serum depleted for 24 h before experiments. Initial dose-response experiments ranging from 100 nM to 10 µM metastin concentrations were performed to determine optimal doses for assessment of cell signaling, migration, and proliferation (data not shown). Cell signaling effects of metastin were detected beginning at the 500 nM dose for GPR54-expressing NPA cells, similar to ARO cells. Growth inhibition was not detected until incubation with 1 µM metastin. Thus, 500 nM metastin was used for migration experiments, and 1 µM was used for cell growth experiments. Metastin (500 nM) inhibited the ability of GPR54-expressing NPA cells to migrate in comparison with vehicle control (Fig. 3, A and B). No inhibitor response was seen for either wild-type nontransfected NPA cells or vector-only controls. Metastin (1 µM) inhibited the growth of the GPR54-expressing NPA cells (Fig. 3C) but had no effect on null-transfected cells (clone 13) or on wild-type NPA cells. The migration and growth inhibition by metastin were significant (P < 0.001) after quantitation.
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Because the pathways activated by GPR54 are known to be mitogenic and/or tumorigenic for thyrocytes (14, 15), we objectively identified metastin-regulated genes in thyroid cancer cells using cDNA microarrays. Total RNA was isolated from GPR54-expressing NPA cells (clone 19) at baseline and after 1 and 24 h of incubation with recombinant metastin (1 µM) on two separate occasions. These samples were then subjected to a microarray analysis, which was performed in duplicate. To maximize the specificity of the results, only genes that were more than 2-fold different after metastin stimulation in all replicates of both experiments were considered to be regulated by metastin. Using these criteria, five genes were up-regulated after 1 h of metastin stimulation in comparison with baseline. After 24 h of metastin incubation, five genes were down-regulated and 14 genes up-regulated (Table 1).
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Metastin chronically reduces calcineurin activity in thyroid cancer cells
To determine the effect of metastin on calcineurin activity in GPR54-expressing thyroid cancer cells, clone 19 cells were incubated with metastin or vehicle for 24 or 48 h. After an initial increase in calcineurin activity after 30–60 min of incubation, metastin induced an approximately 75% reduction of the calcineurin activity (Fig. 4A) (P < 0.001) at 24 and 48 h. Metastin stimulation had no effect on the calcineurin activity of clone 13 that expresses vector alone. Because expression of the calcineurin-regulated isoform of MCIP-1 (exons 4–7) is positively regulated by NFAT transcription factors and metastin acutely increased calcineurin activity before inducing a prolonged decrease in activity, we evaluated NFAT transcriptional activity using an NFAT-binding/luciferase assay in clone 13 and 19 cells before and after acute metastin stimulation (Fig. 4B). After controlling for transfection efficiency; a 5-fold induction of NFAT-induced luciferase activity was detected in clone 19 cells only (P < 0.05) after 6 h of metastin stimulation, confirming that metastin acutely increases NFAT transcriptional activity consistent with the effects on MCIP-1 expression. In addition, we further demonstrated that pharmacological inhibition of calcineurin activity using cyclosporin A reduced the migration of both clone 13 control and clone 19 NPA thyroid cancer cells at a dose, 2.5 µg/ml (Fig. 5A), that had no effect on cell number in preliminary experiments. Finally, in GPR54-expressing clone 19 cells, the combination of cyclosporin A and metastin (500 nM) was more effective than metastin alone in blocking migration (Fig. 5B), consistent with cooperative interactions.
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To determine whether the level of MCIP-1 protein expression was increased by metastin in GPR54-expressing cells, we performed Western blots and immunofluorescence experiments using an MCIP-1 antibody raised against the same region (exon 4) as the microarray and RT-PCR probes before and after metastin incubation. After 24 h, 1 µM metastin increased levels of MCIP-1 in clone 19 cells (Fig. 6A). There was no similar increase in GPR54-null NPA cells (data not shown). This same antibody was then used to evaluate the expression pattern of MCIP-1 in 10 papillary thyroid cancers and adjacent normal tissue. Of these cases, six had metastasis to regional cervical lymph nodes, including five from which metastatic tumor was available for analysis. Immunoactive MCIP-1 was not detected in any of the normal thyroid tissue samples; these samples therefore served as negative controls, in addition to controls lacking primary antibody. In contrast, MCIP-1 expression was detected in seven of 10 primary papillary thyroid cancers, including three of four without regional metastasis and four of six with regional metastasis. Most importantly, none of the five evaluated nodal metastases expressed MCIP-1, regardless of the MCIP expression profile of the primary tumor (Fig. 6B). The four cases with nodal metastasis that expressed MCIP-1 in the primary tumor were characterized by a MCIP-1 expression pattern in which the regions of the primary tumors devoid of MCIP-1 displayed histological patterns that were different from the MCIP-1-expressing regions but were similar to the MCIP-1-negative metastatic tissue (Fig. 6B). The cells in these regions retained typical cytological features of papillary thyroid cancer that were also seen in the metastatic tissue. The observed loss of expression of MCIP-1 in the metastatic tissue is consistent with a metastasis suppression, because loss of metastasis suppressor expression is typically a tumor-progression rather than a tumor-formation-related event.
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Discussion |
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In the present study, we have extended our previous observations by using the GPR54-null NPA papillary thyroid cancer cell line to create a model to identify genes regulated by metastin in cancer cells. NPA cells were chosen because they do not express endogenous GPR54, they migrate in Boyden chamber assays (17), and they are derived from a papillary cancer, the most common form of thyroid cancer. We initially determined that this overexpressed receptor signals through PKC, p42/44 MAPK, and Akt, similar to endogenous GPR54 in ARO thyroid cancer cells (13). In addition, we demonstrated that recombinant metastin can specifically inhibit the migration of GPR54-expressing NPA thyroid cancer cells. Studies using specific blockers of phosphatidylinositol-3-kinase and MAPK did not alter metastin-mediated metastasis inhibition (unpublished data).
Because the PKC, p42/44 MAPK, and Akt pathways are implicated as mediators of thyroid carcinogenesis and progression (14, 15), and blocking experiments did not demonstrate a role in the antimigration function of GPR54, we used a genomics approach to objectively identify mRNAs that were regulated by metastin in thyroid cancer cells. Genes encoding inhibitors of Akt and calcineurin, TRB3 and MCIP-1 (exons 4–7), respectively, were identified. These results were confirmed by real-time quantitative RT-PCR and Western blot (MCIP-1 only). Because metastin itself increases Akt activity in thyroid cancer cells and inhibition of phosphatidylinositol-3-kinase did not alter metastin-mediated migration inhibition, the functional role of TRB3 up-regulation is uncertain. Therefore, we focused our studies on MCIP-1 (exons 4–7).
The gene encoding MCIP-1, DSCR1, contains four alternative transcriptional start sites resulting in expression of four isoforms with alternative first exons (1, 2, 3, 4) and three common downstream exons (5, 6, 7). Of these isoforms, only expression of the transcript composed of exons 4–7 is positively regulated by calcineurin/NFAT signaling. After enhanced expression of MCIP-1 (4, 5, 6, 7) by NFAT transcription factors in response to calcineurin activation, MCIP-1 (4, 5, 6, 7) protein inhibits calcineurin activity, creating a negative feedback loop (23, 32). The cDNA microarray and real-time RT-PCR probes, and the antibody used for Western blot and immunohistochemistry, recognize only this specific MCIP-1 isoform.
Because of the negative feedback loop, if MCIP-1 (4, 5, 6, 7) were functionally involved in this process, one would predict a biphasic calcineurin activity response to metastin stimulation. Indeed, in GPR54-expressing NPA cells, after an initial activation after 30–60 min, metastin reduced calcineurin activity at 24 and 48 h using a well-validated calcineurin assay (31). This inhibitory response occurred in concert with the increase in MCIP-1 protein levels. In further support of a role for this feedback loop in GPR54 effects, we demonstrate that metastin treatment acutely induced NFAT transcriptional activity in luciferase assays, consistent with the initial increase in MCIP-1 expression. The potential role for NFAT-regulated genes other than MCIP-1 (4, 5, 6, 7) in the metastin effect is being investigated.
Recently published data have demonstrated the ability of MCIP-1 to block vascular endothelial growth factor-induced cell motility and angiogenesis in vascular endothelial cells via calcineurin inhibition (33, 34, 35). In the present study, we demonstrated a functional role for the calcineurin pathway in NPA thyroid cancer cell migration by the inhibitory effect of cyclosporin A (Fig. 5
). Because cyclosporin A is not entirely specific for calcineurin, cells were treated with a specific peptide inhibitor of calcineurin A-NFAT binding (VIVIT). This agent also inhibited NPA cell migration (Espinosa, A. V., and M. D. Ringel, unpublished data). The precise role of MCIP-1 as a regulator of thyroid cancer cell migration and the mechanisms responsible for its persistent expression at the protein level in thyroid cancer cells are areas of active investigation.
The clinical association data support a potential functional role for MCIP-1 in metastasis suppression in thyroid cancer. The majority of the examined papillary thyroid cancers express MCIP-1, and this expression was lost in the nodal metastasis, a pattern consistent with loss of expression during tumor progression similar to other metastasis suppressors. Interestingly, the regions of the primary cancer with histological similarity to the metastasis do not express MCIP-1 (Fig. 6). Because of the importance of comparing normal, primary tumor, and metastatic tissue samples from the same patient, we have focused the initial clinical correlation study on papillary thyroid cancers from whom these tissues are most readily available. We are planning to analyze similar sets of tissues from patients with distant metastasis from papillary cancer and/or follicular cancer as soon as they are available, particularly in light of our previous study demonstrating lower levels of GPR54 mRNA levels in primary follicular thyroid cancers (13). Additional studies evaluating the relationship between expression of metastin, GPR54, NFAT, and MCIP-1 in clinical samples are ongoing.
Calcineurin activity has not been assessed in thyroid cancer and is only minimally studied in other malignancies. However, calcineurin activity is essential for neutrophil migratory response to chemoattractants (22), and inhibitors of calcineurin, such as cyclosporin A and FK506, have been used as immunosuppressive therapy after solid organ transplantation. Lymphoid malignancies are more common after organ transplantation, and the incidence of some solid malignancies is also increased (36). However, whether or not these cancers display a reduced tendency toward metastatic spread or are identified because of increased surveillance remain uncertain.
In summary, we have created a novel model of functional GPR54 overexpression in human papillary thyroid cancer cells. Activation of GPR54 inhibits thyroid cancer cell growth and motility in vitro and activates PKC, p42/44 MAPK, and Akt. Using cDNA microarray, we have identified, and confirmed, that the metastin/GPR54 increases mRNA levels of the DSCR1 gene and its protein product, MCIP-1, an inhibitor of calcineurin. Expression of MCIP-1 is increased in the majority of the examined primary papillary thyroid cancers but is lost in lymph node metastasis, consistent with a potential metastasis-suppressing role. Finally, we have also demonstrated that the metastin/GPR54 pathway chronically inhibits calcineurin activity in NPA thyroid cancer cells and that inhibition of calcineurin inhibits cell motility in vitro. These data suggest a previously unrecognized role for calcineurin in the metastasis-suppressive mechanism of the metastin/GPR54 pathway in cancer and suggest that regulation of this pathway may have therapeutic benefit.
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Footnotes |
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First Published Online July 5, 2005
Abbreviations: Cye3, Cyanine 3-dUTP; Cye5, cyanine 5-dUTP; FCS, fetal calf serum; GPR, G protein-coupled receptor; MCIP-1, myocyte-enriched calcineurin interacting protein 1; NFAT, nuclear factor activated in T-lymphocytes; PKC, protein kinase C.
Received May 2, 2005.
Accepted June 29, 2005.
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