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Regulation of CD97 Protein in Thyroid Carcinoma¹

Klinik für Allgemeinchirurgie (C.H.-V., I.S., G.K., H.D.) and Klinik für Kinderheilkunde (K.B.), Martin Luther Universität Halle-Wittenberg, Halle; and Institut für Anatomie, Universität Leipzig (G.A.), and Abteilung Molekulare Innere Medizin, Medizinische Poliklinik, Universität Würzburg (C.S., J.K.), Würzburg, Germany

Address all correspondence and requests for reprints to: Dr. Cuong Hoang-Vu, AG Experimentelle and Chirurgische Onkologie, Klinik für Allgemeinchirurgie, Martin Luther Universität, Magdeburger Strasse 18, 06097 Halle/S, Germany. E-mail: hoang-vu{at}medizin.uni-halle.de

	Abstract

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CD97 is a dimeric glycoprotein belonging to the secretin receptor superfamily and is abundantly expressed in cells of hematopoietic origin. The aim of this study was to analyze the expression of the CD97 protein in thyroid carcinomas and the role of all-trans-retinoic acid (RA) in the regulation of CD97 protein in monolayer culture of the human follicular thyroid carcinoma cell line FTC-133. In normal thyroid tissue, no immunoreactivity of CD97 could be found, whereas in differentiated thyroid carcinomas, CD97 expression was either lacking or low. Undifferentiated anaplastic thyroid carcinomas revealed high CD97 expression. The expression of CD97 protein seems to be correlated to the postoperative histopathological classification staging. Approximately 50% of FTC-133 cells expressed the CD97 protein under basal culture conditions. No differences were found in the number of CD97-positive cells after TSH, forskolin, and insulin treatment compared to control values. Epidermal growth factor treatment led to an increase in CD97 immunostaining (up to 90%), whereas phorbol 12-myristate 13-acetate slightly decreased the immunoreactivity of CD97 (from 50% to 30%). Under basal conditions, RA treatment for 72 h led to a decrease in total cell number by 33% and in CD97-positive cells from 50% to 30%. TSH, forskolin, phorbol 12-myristate 13-acetate, and insulin showed no effect after 72-h pretreatment with RA, whereas epidermal growth factor treatment led to a slight increase in the number of the CD97-positive cells (from 30% to 40%) compared to the control value. These data suggest that CD97 expression may play an important role in the dedifferentiation of thyroid tumors, and RA might interfere with this process in thyroid carcinoma by suppressing the dedifferentiation marker CD97.

	Introduction

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CD97 IS KNOWN as a leukocyte-restricted, cell surface glycoprotein expressed constitutively by human granulocytes, monocytes, and, at low levels, resting T and B cells. Activation of lymphocytes by various stimuli causes a rapid increase in CD97 antigen expression within a few hours (1). CD97 has a novel structure for a seven-transmembrane receptor; it consists of two noncovalently associated subunits, a large extracellular domain (CD97), which occurs or appears in three alternatively spliced isoforms containing between three and five epidermal growth factor (EGF)-like repeats, and CD97ß, a G protein-coupled seven-transmembrane hormone receptor (2, 3, 4). It has been demonstrated that CD97 has features of a multifunctional protein that may play a role in signal transduction associated with the development or establishment of the inflammatory processes (4). Semiquantitative RT-PCR showed a correlation of CD97 messenger ribonucleic acid (mRNA) and cell surface CD97 protein expression levels in several thyroid carcinoma cell lines (5). These distinct structural elements suggest a dual ligand interaction for CD97: 1) binding to extracellular matrix components or other cell surface molecules via the EGF-like domains, and 2) a hormone receptor function mediated by the seven-span transmembrane region.

TSH is a glycoprotein consisting of two subunits, which binds TSH receptor and activates the adenylate cyclase and phospholipase C pathways. TSH receptor belongs to a class of G protein-coupled cell membrane receptors that have extracellular domains that bind the ligand, intracellular domains that activate G proteins, and a transmembrane region that links the two domains and traverses the cell membrane seven times (seven-span transmembrane region) (6; for review, see Ref. 7).

All-trans retinoic acid (RA), an agent with well characterized differentiation-inducing properties, is able to induce intercellular adhesion molecule-1 (8), alkaline phosphatase, and type 1 iodothyronine-5'-deiodinase in thyroid carcinoma cells (9). The redifferentiating effects of RA are confined to at least partly differentiated thyroid cancers and are not seen in anaplastic thyroid cancer (10). In vitro experiments have shown that RA down-regulates sodium iodide symporter (NIS) mRNA and iodide uptake in normal, nontransformed thyrocytes of rats, but up-regulates NIS mRNA in the human follicular thyroid carcinoma cell line FTC-133 (11). In some patients with nonaccumulating thyroid tumors or metastases, RA induces iodide uptake (12).

Several models have been proposed to understand the evolution of cell and tumor biology of thyroid cancer. To date, three different forms of malignant thyroid cancer (follicular, papillary, and anaplastic form) can be distinguished that overlap in part with respect to their tumor markers or pathogenesis. Current treatment protocols using radioiodine, surgical thyroidectomy, or TSH-suppressive T₄ therapy fail in a considerable number of thyroid cancers, and external radiation and chemotherapy are rather ineffective compared to their effects on other cancers.

The aim of the present investigation was to study whether CD97 expression would serve as a marker of dedifferentiation in human thyroid tumors and to study the effects of factors regulating thyroid growth and differentiation, such as TSH, forskolin, phorbol 12-myristate 13-acetate (PMA), EGF, and insulin, as well as RA for the expression of CD97 protein in monolayers of the human follicular thyroid carcinoma cell line FTC-133.

	Materials and Methods

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Ethical approval

The studies involving the use of human tissues described in this paper were approved by the local committees of medical ethics, and all patients gave written consent.

Thyroid tissues

Thyroid tissue was obtained from patients undergoing surgery for thyroid carcinomas (differentiated, n = 15; undifferentiated, n = 14). In one patient we studied the normal thyroid tissue, the primary tumor, and a lymph node metastasis. Tissues from benign diffuse goiter and normal thyroid tissue obtained at surgery for primary hyperparathyroidism (n = 12) served as a control. Histological diagnoses and the clinical classification (TNM) including tumor stages (T), regional lymph node metastases (N), and distant metastases (M) at the time of surgery, as well as postoperative histopathological classification (pTNM) were determined for each specimen by the Institute of Pathology, Martin Luther Universität (Halle, Germany).

Cell culture and treatments

The human follicular thyroid carcinoma cell line FTC-133, supplied by P. Goretzki (Düsseldorf, Germany), was established from the primary tumor of a follicular thyroid carcinoma, characterized by expressing human thyroglobulin (13). Cells were cultured in DMEM-Ham’s F-12 medium and 10% FCS and grown on coverslips for direct immunostaining and microscopy. Medium was changed every 3–4 days. The confluent cells were cultured in serum-free medium for 24 h, then cells were incubated with or without 1 µmol/L RA (Sigma Chemical Co., St. Louis, MO) for 72 h. After 24 h in RA- and serum-free medium, cells were stimulated with recombinant human TSH (100 µU/mL; Genzyme, Cambridge, MA), forskolin (10^-5 mol/L), EGF (5 ng/mL; Sigma Chemical Co.), PMA (10 ng/mL; Sigma Chemical Co.), or insulin (5 ng/mL; Sigma Chemical Co.) for 72 h.

CD97 immunostaining and evaluation

Surgical specimens of thyroid tissues were snap-frozen in liquid nitrogen and stored at -80 C. Frozen sections were cut at 7 µm and fixed in ice-cold methanol for 10 min followed by a short rinse in 0.2 mol/L PBS. Coverslips and frozen sections were prepared for immunohistochemical staining using the monoclonal antibody BL-Ac/F2 after incubation sequences as described previously (14). The specificity of the immunostaining was checked by omission of single steps in the immunochemical protocol and replacement of the primary antibody with nonimmune serum.

The immunoreactivity was evaluated semiquantitatively using a light microscope (Axioplan, Zeiss, Jena, Germany) by four independent investigators (C.H.-V., K.B., G.K., and I.S.) who were blinded to the histological results. Four or five sections of tumor tissue from each patient and four coverslips from each treatment (see Cell culture and treatment) were stained at the same time. Tissue from a patient with a dedifferentiated thyroid tumor was used for each staining procedure as a positive control. CD97 immunoreactivities of tumor tissue and tumor cells were compared to the positive control values, and the number of positive cells was evaluated, ranging from 0 to +++ (+, <30% positive cells; ++, 30–70% positive cells; +++, >70% positive cells).

Cell proliferation test with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

Five thousand cells were cultured with DMEM-Ham’s F-12 medium in 96-well plates. After 24 h, cells were incubated with or without RA for 72 h, followed by a 72-h stimulation with TSH, EGF, forskolin, PMA, and insulin in the absence of RA. Cells were then stained with MTT for 2 h at 37 C and afterward incubated for 30 min with 40 mmol/L HCl in isopropanol at 37 C. A colored formazan salt develops in the mitochondria of living cells. Optical density was measured at 540 nm with an Elisa reader (SLT, Germany). All determinations were performed in quadruplicate.

Statistical analysis

The differences in the number of positive stained cells were tested for significance by the proportional t test, and the differences between groups in the MTT test were determined using the Mann-Whitney-Wilcoxon test (U test).

	Results

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CD97 protein in thyroid carcinoma tissue

In all normal thyroid tissues, no immunoreactivity of CD97 was found. In differentiated thyroid tumors, the CD97 antigen was not detected in 6, was expressed weakly in 7, and was expressed moderately in 2 tumors (Fig. 1 and Table 1). In 13 of 14 undifferentiated thyroid tumors, the immunostaining of CD97 was strongly positive (Fig. 1 and Table 1). However, in one undifferentiated anaplastic thyroid tumor, immunostaining for CD97 was not detected, and Tg was weakly expressed (data not shown). All six of the CD97-negative differentiated tumors were identified as pT1 (tumor diameter <1 cm), 2 with weakly positive staining (+) as pT2 (tumor diameters between 1–4 cm). All anaplastic primary tumors were classified as pT3–4 tumors (tumor diameter >4 cm or cervical tumor invasion). The CD97 immunoreactivity increased with progression to the pT3–pT4 stage (Fig. 2). Patients with weakly positive or negative CD97 immunoreactivity had neither nodal nor distant metastases and were classified as N0/M0 (n = 14). Distant metastases were present in 1 patient, and nodal metastases were present in 4 patients with strong staining for CD97 (+++, M1 and N1, respectively). In the patient with available primary tumor tissue, a lymph node metastasis, and adjacent normal thyroid tissue, the CD97 immunoreactivity was almost undetectable in the normal thyroid, but was strongly positive in the primary tumor and the nodal metastases (Fig. 3).

View larger version (120K): [in this window] [in a new window]   Figure 1. Immunoreactivity for CD97 (original magnification, x40). A, Papillary thyroid carcinoma tissue with weakly to moderately expressed CD97 (30% positive stained cells or ++). B, Weakly expressed CD97 of a follicular thyroid carcinoma (<30% or +). C, Undifferentiated anaplastic thyroid carcinoma with strongly expressed CD97 (>70% positively stained cells).

View larger version (42K): [in this window] [in a new window]   Figure 2. Semiquantitative evaluation of CD97 immunoreactivity in correlation with pTNM stages. The stages of thyroid differentiation were determined after TNM/pTNM classification. CD97 negative, 0–30% positive cells. CD97 positive, more than 30% positive cells.

View larger version (113K): [in this window] [in a new window]   Figure 3. Immunoreactivity for CD97 of thyroid tissues and nodal metastasis of the same patient. A, Normal thyroid tissue; CD97 immunoreactivity was found in only some thyrocytes (evaluated as 0). B, primary tumor tissue strongly stained (evaluated as +++). C, Lymph node metastasis strongly stained.

Approximately 50% of FTC-133 cells expressed the CD97 protein under basal culture conditions. No differences were found in CD97 immunoreactivity after TSH, forskolin, and insulin treatment compared to control values. EGF treatment led to a significant increase in CD97 immunostaining (90%), whereas PMA slightly decreased the immunoreactivity of CD97 (30%; Fig. 4).

View larger version (113K): [in this window] [in a new window]   Figure 4. Immunoreactivity for CD97 of the tumor cell line FTC-133. A, Control; B, PMA treatment for 24 h; C, TSH treatment for 24 h; D, EGF treatment for 24 h.

MTT test evaluation revealed that RA treatment led to a decrease in the total cell number of about 18% after 24 h, 25% after 48 h (P < 0.05), and 33% after 72 h (P < 0.01) compared with the untreated control value. The number of CD97-positive cells was not changed after 24-h RA treatment, but decreased from 50% to 42% after 48 h and to 30% after 72 h (P < 0.01) under basal conditions. TSH, forskolin, PMA, and insulin showed no additional effect after pretreatment with RA during the experimental period, whereas EGF treatment led to an insignificant increase in the number of positive cells (from 30% to 40%) after 72-h pretreatment with RA compared to the control value (Fig. 5).

View larger version (118K): [in this window] [in a new window]   Figure 5. Immunoreactivity for CD97 of the tumor cell line FTC-133. A, Control; B, PMA treatment for 24 h; C, TSH treatment for 24 h; D, EGF treatment for 24 h (after pretreatment with 1 µmol/L RA for 72 h).

	Discussion

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Concerning the structure of CD97, the seven-transmembrane region classifies CD97 within the secretin receptor superfamily. The extracellular region of CD97 contains EGF-like domains and a single RDG motif, which is known as a potential recognition sequence of integrins (2) and thereby possesses structural similarities to extracellular matrix proteins. It can be speculated that the dual structure and function of CD97 facilitate the adhesion and simultaneously contribute to a growth advantage of the cancer cells. CD97 might mediate tumor cell attachment, whereas activation of cAMP and/or protein kinase C pathways through the seven-transmembrane receptor domain might promote deregulated growth and clonal expansion of the thyroid carcinoma cells. Our data showed that protein kinase C activation by PMA via the phospholipase C signaling pathway led to down-regulation of CD97 protein expression in thyroid tumor cells. There might exist an autoregulation loop between PKC activation and CD97 expression.

The agents that influenced CD97 expression on the cell surface in vitro were EGF and PMA. In contrast, TSH and forskolin as well as insulin, which have been shown to affect a multitude of cellular functions in human thyrocytes and carcinoma cell lines (15), had no effect on CD97 expression. Our data are further supported by our previous finding that EGF, which stimulates the growth and invasion of differentiated thyroid carcinoma cells (15), also up-regulated CD97 in these cells (16). The reduction of cell number after RA stimulation for 72 h might indicate the induction of apoptosis and/or necrosis in this cell line. In acute promyelocytic leukemia, Tomizuka et al. have shown that apoptosis-related genes, such as CD95 (Fas/Apo-1), are stimulated by RA treatment in vivo, and fresh acute promyelocytic leukemia cells expressed CD95 after the addition of RA in vitro (17). Our preliminary data showed that RA treatment induced the expression of CD95 protein by stimulating its translation in the follicular thyroid carcinoma cell line FTC-236 (18). RA not only acts on the Fas/Fas ligand system but also induces iodide uptake in patients with nonaccumulating thyroid tumors (12) and up-regulates NIS mRNA in the human follicular thyroid carcinoma cell line FTC-133 in vitro (11). In contrast, RA down-regulates NIS mRNA and iodide uptake in normal, nontransformed thyrocytes of rats (11), but RA also induces retinoid receptors and alters the pattern of RA receptor subtypes expressed along with modulation of several other RA-responsive gene products (19). Therefore, putative ligand-dependent signaling by CD97 may convey additional regulatory pathways to transformed thyrocytes, which frequently lose control by the thyroid-specific TSH receptor-adenylate cyclase signal transduction pathway. Expression of TSH receptor is lost in many thyroid tumors (20), which then might not respond to TSH-suppressive T₄ therapy. Therefore, RA could be useful for a differentiation therapy protocol in selected thyroid carcinoma not responding to T₄ therapy or lacking radioiodide uptake.

In summary, our data, obtained from a limited number of differentiated and undifferentiated thyroid carcinomas, have shown that strong immunostaining for CD-97 was associated with the aggressiveness and lymph node involvement of these tumors. The effects of RA treatment of follicular thyroid carcinoma cells, FTC-133, suggest that RA might be able to modulate invasiveness and aggressiveness in thyroid carcinoma by inducing the expression of differentiation markers and by suppressing dedifferentiation markers, such as CD97.

	Acknowledgments

 
We thank Mrs. K. Hammje, J. Meissner-Weigl, and M. Sitte for skillful assistance; Prof. Dr. H.-J. Holzhausen, Institute of Pathology, for the histopathological diagnosis of the investigated tissues; and Dr. O. Gimm, Dana-Farber Cancer Institute, for the careful review of the manuscript.

	Footnotes

 
¹ This work was supported by Grant 2792A/0087H from Land Sachsen-Anhalt and by Deutsche Forschungsgemeinschaft (Wi 231/9–2).

Received September 22, 1998.

Revised December 10, 1998.

Accepted December 17, 1998.

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