Thymosin ß-10 gene expression as a possible tool in diagnosis of thyroid neoplasias

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1 ONCOLOGY REPORTS 12: , Thymosin ß-10 gene expression as a possible tool in diagnosis of thyroid neoplasias GENNARO CHIAPPETTA 1, FRANCESCA PENTIMALLI 2, MARIO MONACO 1, MONICA FEDELE 2, ROSA PASQUINELLI 1, GIOVANNA MARIA PIERANTONI 2, MARIA TERESA RIBECCO 1, GIOVANNI SANTELLI 1, DANIELA CALIFANO 1, LUCIANO PEZZULLO 1 and ALFREDO FUSCO 2 1 Istituto dei Tumori di Napoli; 2 Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facoltà di Medicina e Chirurgia, Università di Napoli Federico II, via Pansini 5, I Napoli, Italy Received December 15, 2003; Accepted February 9, 2004 Abstract. Overexpression of thymosin ß-10 (TB10) has been shown in rat thyroid transformed cell lines, and in human thyroid carcinoma tissues and cell lines. To investigate whether TB10 detection could be a valid tool in the diagnosis of human thyroid neoplasias, we extended the analysis of TB10 expression to a large number of thyroid hyperproliferative and neoplastic tissues using an immunohistochemical assay. Our analyses showed a TB10 positive staining in all human thyroid carcinomas particularly in the anaplastic histotypes, whereas no TB10 immunostaining was observed in normal thyroid, in adenomas and the majority of the goiters. These results suggest that the evaluation of TB10 gene expression may be considered a promising means of diagnosis of human thyroid hyperproliferative disorders. Introduction Thymosins comprise a family of small proteins originally isolated from calf thymus (1), and divided into 3 classes (, ß, Á) based on their isoelectric point. The ß class comprises structurally related, highly conserved acidic polypeptides. Thymosin ß-10 (TB10) is a 5 kda polypeptide comprised of 44 amino acid residues (2) and, in human tissues, is widely distributed along with another component of the class, the thymosin ß-4 (TB4) (3). Both proteins have been shown to bind to and sequester G-actin. Moreover, both TB10 and TB4 selectively induce an actin depolymerization resulting in the disruption of intracellular F-actin networks (4,5). Recently, we have demonstrated the overexpression of TB10 in rat thyroid transformed cell lines, and in human thyroid carcinoma tissues and cell lines. TB10 expression Correspondence to: Dr Alfredo Fusco, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facoltà di Medicina e Chirurgia, Università di Napoli Federico II, via Pansini 5, I Napoli, Italy afusco@napoli.com Key words: thymosin, neoplasia, thyroid, immunohistochemistry was particularly abundant in the undifferentiated thyroid carcinomas (6). Moreover, we have shown that thymosin ß- 10 protein synthesis suppression reduces the growth of human thyroid carcinoma cells in semisolid medium (7). We have also shown that TB10 overexpression occurs in several non-thyroid neoplasias such as colon carcinomas, germ cell tumours of different histological types, ovarian carcinomas, uterine carcinomas whereas it was weakly detected in the respective normal tissues (8). Other studies have shown elevated levels of TB10 in melanoma cell lines and human breast cancer, and suggested that its expression level may be used as a progression marker for these tumours (9,10). TB10 upregulation was also demonstrated previously in renal cell carcinoma (8) and recently in disseminated neuroblastomas (10). Furthermore an immunohistochemical assay for TB10 detection was able to reveal TB10 protein expression in breast, colon and ovary carcinoma samples but not in the respective normal tissues (8). Therefore, the TB10 detection may be considered as a potential tool in the diagnosis of several human neoplasias. Thyroid nodules are a common occurrence in the general population, they may be represented by hyperplastic or neoplastic proliferative lesions of thyroid follicular epithelium (13). In the management of patients with solitary thyroid nodules the search for thyroid carcinoma is fully justified since the majority of thyroid carcinoma can be effectively treated. However, thyroid malignant nodules are in objectively difficulty to be identified, and this has induced several research group to the search for tumour markers and prognostic indicators. Our previous studies on thymosin expression in thyroid carcinoma suggested the detection of this protein as a good candidate. Therefore, we extended the analysis of TB10 detection to a large number of human thyroid proliferative disorders. Here we report that TB10 is overexpressed in all human thyroid carcinomas, particularly in the anaplastic histotypes, whereas TB10 immunostaining was absent in normal thyroid, in adenomas and the majority of the goiters. Materials and methods Human thyroid tissues. Normal and pathological thyroid neoplastic samples were collected at the Istituto Nazionale

2 240 CHIAPPETTA et al: THYMOSIN ß-10 EXPRESSION IN THYROID CARCINOMAS Table I. TB10 protein expression in human thyroid tissues. Histological type of No. of positive cases No. of negative cases No. of positive cases/ thyroid samples analysed by immuno- analysed by immuno- no. of total cases analysed histochemistry histochemistry by immunohistochemistry Normal thyroid 0 3 0/3 Goiters /24 Adenomas /39 Microcarcinomas 3 0 3/3 Follicular carcinomas /10 Papillary carcinomas /14 Anaplastic carcinomas 6 0 6/6 dei Tumori di Napoli, Naples, Italy. Tissue samples from thyroid tumours or normal thyroid tissues in the opposite lobe of carcinomas were obtained by surgery. The criteria for inclusion in the study were the ability of routinely processed paraffin blocks for immunohistochemistry and adequate clinical information. Immunohistochemistry. Paraffin sections (5-6 µm) were deparaffinized and placed in a solution of absolute methanol and 0.3% hydrogen peroxide for 30 min and washed first in distilled water, then in phosphate buffer solution (PBS), before immunoperoxidase staining. The slides were then incubated overnight at 4 C in a humidified chamber with the antibodies diluted 1:100 in PBS. The slides were subsequently incubated with biotinylated goat anti-rabbit IgG for 20 min (Vectostain ABC kits, Vector Laboratories) and then with premixed reagent ABC (Vector) for 20 min. For immunostaining, the slides were incubated in diaminobenzidine (DAB-Dako) solution containing 0.06 mm DAB and 2 mm hydrogen peroxidase in 0.05% PBS ph 7.6 for 5 min, and after chromogen development, the slides were washed, hematoxylin counterstained, dehydrated with alcohol and xylene, and mounted with coverslips using a permanent mounting medium (Permount). Micrographs were taken on Kodak Ektachrome film with a photo Zeiss system. The antibodies used in this study, were raised against the synthetic peptide TIEQEKRSEIS specific for the TB10 protein (8) and affinity purified against the same synthetic peptide. Results Immunohistochemical analysis of TB10 expression in thyroid samples. Immunohistochemical analysis allows a rapid and sensitive screening of thyroid pathological tissues and is amenable to regular use as a routine diagnostic test. Therefore, we have analysed for TB10 expression 100 thyroid samples, including 3 normal thyroids, 39 adenomas, 24 goiters, 3 microcarcinomas, 10 follicular, 14 papillary and 6 anaplastic carcinomas. The results of this study are summarized in Table I. All normal thyroid and adenoma samples were negative, whereas all malignant thyroid neoplasias were positive for TB10 expression. Only 5 out of 24 goiters showed TB10 expression. Some representative results of this analysis are shown in Fig. 1. No staining was observed in normal thyroid tissue (Fig. 1A). Conversely, an intense cytoplasmic staining was clearly observed in a papillary carcinoma (Fig. 1B) and in a follicular carcinoma (Fig. 1F). A greater proportion of neoplastic cells positive for TB10 as well as a stronger intensity of the immunohistochemical reaction was generally observed in the undifferentiated (anaplastic), compared to the well differentiated carcinomas (papillary or follicular) (Fig. 1G and H). In Fig. 1E we show a follicular adenoma which did not show any significant staining. A goiter showing positive staining is illustrated in Fig. 1D. To validate the specificity of our anti-tb10 antibodies, for each case, sections were stained without the primary antibody and in all cases these controls were negative, and some neoplastic tissues were also analysed after the TB10 specific antibodies had been pre-incubated with the TB10 control peptide. In Fig. 2 we show a representative papillary carcinoma stained with anti-tb10 antibodies (panel A), in absence of the primary antibody (panel B), and with TB10 specific antibodies preincubated with the TB10 control peptide (panel C). No staining was observed in Fig. 2B and C. Discussion TB10 overexpression is a general event in the process of carcinogenesis. In fact, apart from a report showing TB10 down-regulation in 4 out 5 ovarian carcinomas (11), high TB10 mrna levels have been detected in several human neoplasias (6-12). The aim of our work was to analyse a large number of thyroid neoplastic samples to verify whether TB10 expression might have been used as a tool for the diagnosis of thyroid neoplastic disease. Here we show that all the carcinomas were positive, whereas no positivity was found in adenoma, and just few goiters were positive. This would indicate TB10 detection as a possible tool for differential diagnosis between follicular adenomas and follicular carcinomas, which represents a dilemma for the pathologist. To this purpose, TB10 analysis might be associated to galectin 3 expression which has been recently demonstrated to discriminate between benign and malignant follicular carcinomas (15). The increased expression of TB10 in goiters may be dependent on the TSH induction of TB10 as already shown

3 ONCOLOGY REPORTS 12: , Figure 1. Immunohistochemical analysis of TB10 in thyroid tissues. Paraffin sections from normal and pathological thyroid samples were analyzed by immunohistochemistry using antibodies raised against a specific TB10 peptide. A, Immunostaining of a normal thyroid tissue (x200). No immunoreactivity was observed. B, Immunostaining of a papillary thyroid carcinoma (x200). A cytoplasmic staining was observed. C, Immunostaining of a goiter (x200). No immunoreactivity was observed. D, Immunostaining of a goiter (x200). A cytoplasmic staining was observed. E, Immunostaining of a follicular adenoma (x200). No immunoreactivity was observed. F, Immunostaining of a follicular carcinoma (x200). A cytoplasmic positivity was observed. G, Immunostaining of an anplastic thyroid (x200). A very strong cytoplasmic staining was observed. H, Immunostaining of an anplastic thyroid (x400). A very strong cytoplasmic staining was observed.

4 242 CHIAPPETTA et al: THYMOSIN ß-10 EXPRESSION IN THYROID CARCINOMAS the backbone vector (7). A role of TB10 in the progression step of carcinogenesis seems also confirmed by the mouse model of skin carcinogenesis induced by the combined action of chemical carcinogens and phorbol esters. In fact, TB10 expression was almost undetectable in normal keratinocytes, its induction occurred even at the papilloma stage, however a further increased expression was observed in the carcinoma derived cell lines (8). It is likely that the TB10 role in cancer progression might be linked to cell motility that is somehow depending on the assembly of actin filaments. Accordingly it has been shown that TB10 overexpressing stable transfectants derived from a NIH 3T3 subline have thicker filaments, higher motility and faster spreading ability compared with the vector transfected cells (17). Equally, profound overexpression of TB10 caused the almost complete disappearance of stress fibers and the localization of filamentous actin in globular rosette-like structures at the cell periphery in the thyroid carcinoma cell line NPA (7). In conclusion, TB10 overexpression may account for the high invasivity property of the undifferentiated thyroid tumors, and the detection of the TB10 protein may represent a useful tool for the diagnosis of thyroid neoplastic diseases. Acknowledgements This study was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), by the Programma Italia-USA sulla Terapia dei Tumori coordinated by Professor Cesare Peschle, Progetto Finalizzato Biotecnologie of Consiglio Nazionale delle Ricerche, Progetto 5% of the Consiglio Nazionale delle Ricerche, and the MURST Project Terapie antineoplastiche innovative. We are indebted to Jean Gilder for editing the text. References Figure 2. TB10 expression is specific for thyroid neoplastic cells. A, Immunostaining of a papillary carcinoma (x200). A cytoplasmic staining was observed. B, Immunostaining of the same papillary carcinoma as in (A) in the absence of the primary antibodies (x200). No immunoreactivity was observed. C, Immunostaining of the same papillary carcinoma as in (A) a papillary thyroid carcinoma with the TB10 antibodies pre-incubated with the peptide against which antibodies were raised. No immunoreactivity was observed. in rat experimental goiters and in TSH-stimulated thyroid cells (16). Another important point of our studies is represented by the highest level of TB10 expression in anaplastic carcinomas suggesting a TB10 role in the progression step. This is consistent with our previous studies showing that the suppression of TB10 protein synthesis, by an antisense methodology, in two thyroid carcinoma cell lines, NPA and ARO induced a significant reduction in their ability to grow in soft agar, which was not modified by the transfection of a expression vector carrying the TB10 in sense orientation or 1. Low TLK and Goldstein AL: Structure and function of thymosin and other thymic factors. In: The Year in Hematology. Silber R, Lobue J and Gordon AS (eds). Plenum Press, New York, pp , Erickson-Viitamen S, Ruggieri S, Natalini P and Horecker BL: Thymosin beta 10, a new analog of thymosin beta 4 in mammalian tissues. Arch Biochem Biophys 22: , Yu FX, Lin SC, Morrison-Bogorad M and Yin HL: Effects of thymosin beta 4 and thymosin beta 10 on actin structures in living cells. Cell Motil Cytoskeleton 27: 13-25, Sanders MC, Goldstein AL and Wang YL: Thymosin beta 4 (Fx peptide) is a potent regulator of actin polymerization in living cells. Proc Natl Acad Sci USA 89: , Safer D, Elzinga M and Nachmias VT: Thymosin ß4 and Fx, an actin-sequestering peptide, are indistinguishable. J Biol Chem 266: , Califano D, Monaco C, Santelli G, Giuliano A, Veronese ML, Berlingieri MT, De Franciscis V, Berger N, Trapasso F, Santoro M, Viglietto G and Fusco A: Thymosin ß-10 gene overexpression correlated with the highly malignant neoplastic phenotype of transformed thyroid cells in vivo and in vitro. Cancer Res 58: , Santelli G, Bartoli PC, Giuliano A, Porcellini A, Mineo A, Barone MV, Busiello I, Trapasso F, Califano D and Fusco A: Thymosin beta-10 protein synthesis suppression reduces the growth of human thyroid carcinoma cells in semisolid medium. Thyroid 12: , Santelli G, Califano D, Chiappetta G, Vento MT, Bartoli PC, Zullo F, Trapasso F, Viglietto G and Fusco A: Thymosin beta-10 gene overexpression is a general event in human carcinogenesis. Am J Pathol 155: , 1999.

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