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Dipartimento di Scienze e Tecnologie Biomediche and Dipartimento di Scienze Mediche e Morfologiche (A.V.D., F.P., P.C., C.D., G.D.), Università di Udine, 33100 Udine, Italy; Microgravity Aging Training Immobility Center (A.V.D., G.D.), Udine, Italy; Dipartimento di Biofisica, Biochimica e Chimica delle Macromolecole (G.T., G.M.), Università di Trieste, I-34127 Trieste, Italy; Dipartimento di Scienze Farmacobiologiche and Dipartimento di Medicina Sperimentale e Clinica (D.R., F.A., S.F.), Università di Catanzaro, 88100 Catanzaro, Italy; Clinical and Experimental Hematology Research Unit (V.G.), Centro di Riferimento Oncologico, Instituto di Ricovero e Cura a Carattere Scientifico, 33081 Aviano, Italy; Indiana Cancer Research Institute (D.L.M.), Indianapolis, Indiana, IN 46202-5254
Address all correspondence and requests for reprints to: Prof. Giuseppe Damante, Dipartimento di Scienze e Tecnologie Biomediche, Piazzale Kolbe 1-33100 Udine, Italy. E-mail: . GDamante{at}makek.dstb.uniud.it
Abstract
Homeobox genes are involved in neoplastic transformation of both epithelial and hemopoietic tissues. The divergent homeobox gene HEX is expressed in the anterior visceral endoderm during early mouse development and in some adult tissues of endodermal origin, including liver and thyroid. Whereas a role in leukemyogenesis has been proposed already, few data are available on the involvement of HEX in human epithelial tumors. Herein, we analyzed HEX expression and subcellular localization in a series of 55 human thyroid tumors and in several tumoral cell lines. HEX mRNA was detected by RT-PCR either in normal tissues or in thyroid adenomas and differentiated (papillary and follicular) carcinomas. HEX mRNA was also expressed in most undifferentiated carcinomas. Subcellular localization of HEX protein was investigated by immunohistochemistry. In normal tissues and adenomas, HEX protein was present both in nucleus and cytoplasm. In contrast, both differentiated and undifferentiated carcinomas, as well as the tumoral cell lines investigated, showed HEX protein only in the cytoplasm. These findings suggest that regulation of HEX entry in the nucleus of thyrocytes may represent a critical step during human thyroid tumorigenesis.
CONTROL OF GENE expression is mostly exerted at the transcriptional level, through the action of transcription factors. These proteins bind to regulatory sequences of target genes and act by modifying either the basal transcriptional machinery or the chromatin structure (1, 2). Transcription factors can be classified as ubiquitous or tissue-specific. The latter are present only in a few cell types and participate to the control of genes expressed only in particular tissues (3, 4). Among tissue-specific transcription factors, DNA-binding proteins encoded by homeobox genes are included (5). These proteins regulate gene expression during development and may keep playing a role in maintaining the differentiate phenotype in the adult tissue. Because of their crucial role in cell differentiation, alteration of structure or expression of tissue-specific transcription factors is relevant in neoplastic transformation (6).
In the thyroid follicular cell (TFC), several tissue-specific transcriptional regulators have been identified, including TTF-1, TTF-2, PAX8 and HEX (7, 8, 9, 10). These proteins control the transcription of genes expressed predominantly in the thyroid tissue such as Tg, thyroperoxidase (TPO), TSH receptor and sodium/iodide symporter (NIS) genes (11 and references therein). Some of them, in addition, control TFC proliferation (12) and, when overexpressed, may act as oncogenes (13). Expression and functions of TTF-1 and PAX8 have been investigated in both human thyroid tumors and transformed cell lines (14, 15, 16). TTF-1 is always expressed in differentiated thyroid carcinomas. In contrast, its expression is extinguished in most undifferentiated carcinomas (14, 17, 18, 19). Conversely, PAX8 expression is abolished in a significant fraction of differentiated thyroid carcinomas (14, 20). The abolition of PAX8 expression by cell transformation appears as a key event for the loss of differentiation observed in thyroid tumors. In fact, Tg mRNA levels are strictly correlated to PAX8 mRNA levels in papillary carcinomas (21). Moreover, reintroduction of PAX8 in transformed rat thyroid cell lines is sufficient to activate expression of endogenous genes encoding Tg, TPO, and NIS (22). Recent data indicate that a chromosomal translocation involving the PAX8 gene is specific for follicular thyroid carcinomas (23). Altogether, these data indicate that investigations on tissue-specific transcription factors in thyroid tumors may allow a better understanding of mechanisms operating for TFC differentiation and could provide interesting markers for a molecular characterization of thyroid neoplasms. In addition, detailed knowledge on thyroid-specific transcription factors may help in setting up efficient approaches of gene therapy of thyroid tumors. An example of this possibility has been provided recently; cotransduction of TTF-1 together with a chimeric construct in which transcription of a suicide gene (HSV-TK) is controlled by the Tg promoter, confers targeted cytotoxicity in poorly differentiated thyroid carcinoma cells but not in nonthyroid cells (24).
The homeobox gene HEX, also known as Prh (25, 26), is expressed in several hematopoietic progenitors in which it is down-regulated during cell differentiation (27). HEX gene is also expressed in endothelial cells and, during early embryogenesis, in anterior visceral endoderm. During organogenesis, HEX is expressed in several tissues of endodermal origin, including lung, liver, and thyroid (10). In this latter gland, HEX expression appears to be extremely critical. In fact, in the HEX -/- mouse, thyroid development is arrested at the bud stage at 9.5 d post coitum (28). We have previously demonstrated that in TFC, HEX is expressed up to the adult stage in which it down-regulates the Tg gene expression, counteracting the effects of TTF-1 and Pax8 at the Tg promoter level (29). Moreover, HEX expression was completely abolished in some rat thyroid cell lines transformed by oncogenes (29).
In the present study, we examined the expression of HEX and its cellular localization in a series of human thyroid tumors, including both benign and malignant neoplasia, and in four human thyroid tumoral cell lines.
Materials and Methods
Thyroid tissue samples
Fifty-five samples of thyroid tissue were obtained from patients who had undergone surgical resection of the thyroid gland for benign or malignant lesions. Specimens used to extract RNA were quickly frozen and stored at -80 C or in liquid nitrogen. Tissue samples included 10 toxic adenomas, 10 follicular adenomas, 20 papillary carcinomas, 10 follicular carcinomas, and 5 undifferentiated carcinomas. Histological classification was based on the criteria described by Hedinger et al. (30). The surrounding unaffected tissues from cases of follicular adenomas were considered as representative specimens of normal thyroid tissue. This study was approved by the local ethical committee.
Detection of HEX mRNA by RT-PCR
Total RNA was extracted from frozen fresh tissues by using the RNA B technique (Bioprobe Systems, Richmond, CA), following the manufacturer’s instructions as previously described (31). The tumor tissues used for RNA isolation in this study were microdissected by the pathologist to exclude contamination of surrounding normal thyroid cells. cDNA was synthesized from 1 µg total RNA according to the manufacturer’s instructions (Roche Diagnostics SpA, Monza, Italy) as previously described (32). The mixture was incubated at 25 C for 10 min and 42 C for 60 min, heated to 99 C for 5 min, and then stored at -20 C. PCR amplification was performed using 5 µl of cDNA, as previously described (33). Briefly, the samples were subjected to 30 and 40 cycles of amplification reaction. Amplification of 40 cycles was performed to exclude that HEX-negative samples would produce a small amount of HEX mRNA. PCR conditions for the HEX gene were as follows: denaturation at 94 C for 1 min, annealing at 58 C for 1 min, and extension at 72 C for 1 min. Ten of 50 µl of the amplification products were then run on 1.5% Tris-Borate-EDTA agarose gel containing ethidium bromide and analyzed to confirm a positive or negative outcome. Primer oligonucleotides for the HEX gene were: 5' primer, 5'-TACTCTGGAGCCCCTTC TTG-3', and 3' primer, 5'-TTCAAGGTCTTCCTGGGAGG-3'. The amplification yielded a 371 bp DNA product corresponding to fragment 377–748, according to the published sequence of the HEX gene (25). As control, we used the ß-actin gene and the PCR conditions were as follows: denaturation at 94 C for 1 min, annealing at 62 C for 1 min, and extension at 72 C for 1 min. Primer oligonucleotides for the ß-actin gene, were: 5' primer, 5'-CGAGGCCCAGAGCAAGACA-3', and 3' primer, 5'-CACAGCTTCTCCTTAATGTCA CG-3'. The amplification yielded a 482 bp DNA product corresponding to fragment 209–691, according to the published sequence of the ß-actin gene (34). All primers were from Life Technologies, Inc. [S.r.l., San Giuliano Milanese (Milan), Italy]. mRNAs from HeLa cell line and from medullary carcinomas (no. 4) were used as negative controls.
Immunohistochemistry
Formalin-fixed, paraffin-embedded thyroid tissue samples were evaluated for the expression of HEX protein using an immunoperoxidase technique. The surrounding unaffected tissues from five cases of follicular adenomas were obtained as representative specimens of normal thyroid. Representative blocks of each case were selected for immunohistochemical staining. Sections of representative blocks of thyroid lesions that were formalin-fixed and paraffin-embedded were cut onto silane-coated slides and dewaxed. After blocking of endogenous peroxidase, sections were incubated with rabbit antiserum to HEX diluted 1:250 in PBS overnight at 4 C. After washing, the biotinylated goat antirabbit antibody, at a dilution of 1:200, and the avidin-peroxidase detection system (ABC Elite Vectastain, Vector Laboratories, Burlingame, CA) were both applied for 30 min at room temperature. Peroxidase activity was detected with 3,3'-diaminobenzidine tetrachloride (Sigma, St. Louis, MO) and hematoxylin counterstain was applied. Negative controls were carried out, replacing the primary antiserum with preimmune rabbit serum. The HEX antibody was raised in rabbits against a peptide corresponding to residues 118–135 of the whole HEX protein by using the standard procedures. This antibody was previously used for HEX immunodetection, and its specificity has been demonstrated (35). A brown staining in the nucleus and/or in the cytoplasm indicated the cellular presence of HEX protein. The HEX expression was evaluated by calculating separately nuclear and cytoplasmic reactivity in 1000 cells (identified by hematoxylin counterstain) and was expressed as a percentage of positive cells. In the case of cell cultures (see below), cells were immediately fixed with ethanol containing 5% acetic acid glacial for 10 min at 4 C. Then, cells were rinsed with 0.1% saponin (Sigma) in PBS. This PBS-saponin solution was also used for all subsequent washing steps. The cultures were incubated with rabbit antiserum to HEX diluted 1:100 overnight at 4 C. After washing, biotinylated goat antirabbit antibody at a dilution of 1:200 and the streptavidin-phosphatase alkaline detection system (StreptABComplexes/AP, DAKO Corp., Glostrup, Denmark) were both applied for 30 min at room temperature.
Alkaline phosphatase was developed with new fuchsin (fuchsin + substrate, Chromogen, DAKO Corp.) containing levamisole. Control staining used preimmune rabbit serum (DAKO Corp.) instead of the primary antiserum.
Cell cultures
Human thyroid carcinoma cell lines BCPAP, WRO, FRO, and ARO were used (36, 37). BCPAP, WRO, and FRO cell lines were grown in DMEM supplemented with 10% FBS. The ARO cell line was grown in RPMI 1640 supplemented with 10% FBS. Cell nuclear extracts were prepared as previously described (38). Briefly, 107 cells were washed once with PBS and resuspended in 500 µl of hypotonic lysis buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM MgCl2, 0.1 mM EDTA, 2 µg/ml leupeptin, 2 µg/ml pepstatin, 0.5 mM PMSF, pH 7.9). After 10 min, cells were homogenized by 10 strokes with a loose-fitting Dounce homogenizer. Nuclei were collected by centrifugation for 5 min at 500x g at 4 C in a microcentrifuge. The supernatant obtained after this centrifugation was considered as the cytoplasmic fraction. Nuclear proteins were extracted with 100 µl of buffer B (10 mM HEPES, 400 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 2 µg/ml leupeptin, 2 µg/ml pepstatin, 0.5 mM PMSF, pH 7.9). After incubating for 20 min at 4 C, samples were centrifuged at 12,000x g at 4 C for 15 min. Nuclear and cytoplasmic extracts were then quantitated for protein levels (39) and used immediately for Western blot analysis or kept at -80 C.
Western blot analysis
Thirty micrograms of nuclear or cytoplasmic extracts, obtained from different human thyroid cell lines, were electrophoresed onto a 12% SDS-PAGE. Then, proteins were transferred to nitrocellulose membranes (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH). After transfer, membranes were saturated by incubation at 4 C overnight with 5% nonfat dry milk in PBS/0.1% Tween-20 and then incubated with the anti-HEX rabbit polyclonal antibody for 120 min at room temperature. After three washes with PBS/0.1% Tween-20, membranes were incubated with an antirabbit immunoglobulin coupled to peroxidase (Sigma). After 60 min of incubation at room temperature, the membranes were washed several times with PBS/0.1% Tween-20, and the blots were developed using ECL chemiluminescence procedure (Amersham Pharmacia Biotech, Milan, Italy).
Results
HEX mRNA expression in thyroid tumors
Oncogene-transformed rat thyroid cell lines displayed a loss of HEX gene expression (29). However, the cell lines investigated in that study had a very high degree of dedifferentiation, showing loss of all markers of the thyroid-differentiated phenotype. HEX expression has been evaluated also in another model of epithelial transformation; poorly differentiated hepatoma cells do not express HEX, which, instead, is expressed in highly differentiated hepatoma cells (40). In the present study, we examined the pattern of HEX expression in human thyroid tumoral tissues. The presence of HEX mRNA was evaluated by RT-PCR. Expression of the ß-actin gene was used as amplification control. Both 30- and 40-cycle amplifications were performed in all samples. Forty-cycle amplification was performed to verify that the absence of HEX transcript in negative samples (according to the 30-cycle amplification) would be due to a real absence of HEX mRNA and not to low expression levels. mRNAs from HeLa cell line and from four medullary carcinomas were used as negative controls. All of these samples show absence of HEX mRNA after both 30- and 40-cycle amplifications (data not shown). Results of representative samples after 40-cycle amplification are shown in Fig. 1
. As expected, HEX expression was present in all normal thyroid tissues tested. Among thyroid pathological tissues, HEX transcript was always observed in adenomas and differentiated carcinomas (papillary and follicular) (Fig. 1; data not shown). HEX expression was not observed in one of five cases of undifferentiated carcinomas investigated (Fig. 1; data not shown). Similarly, several human thyroid tumoral cell lines maintained HEX mRNA expression (data not shown). These results indicate that HEX expression is commonly not abolished in human thyroid tumors, delineating an expression pattern different from those of TTF-1 and Pax8. In only in a fraction of undifferentiated carcinomas is HEX expression extinguished, indicating, at least from the molecular point of view, heterogeneity for this tumor type.
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Next, we examined the HEX protein expression in our series of thyroid tumors. Although HEX is known as a transcriptional regulator, in addition to the nuclear localization it exhibits a cytoplasmatic localization in several tissues (41). Thus, by using immunohistochemistry, HEX subcellular localization was evaluated in human thyroid tumors. Immunohistochemical detection of HEX was performed by using a specific antibody that was characterized in a previous study (35). Representative immunohistochemical samples are shown in Fig. 2. Quantitation of positive cells and nuclear/cytoplasmic localization in the various types of thyroid tissues investigated is shown in Fig. 3. All normal and pathological tissues that were examined demonstrated the presence of HEX protein. In normal tissues, adenomas, and differentiated carcinomas, about 10% of the HEX-negative cells were detected. In contrast, undifferentiated carcinomas showed 25% of HEX-negative cells (Fig. 3A). The difference of HEX-negative cells between undifferentiated carcinomas and all other types of thyroid tumors or normal tissue is significant, according to the t test (P < 0.05 in all cases). As expected, in normal tissues HEX protein was present in both nuclear and cytoplasmatic compartments. Both the nuclear and the cytoplasmatic staining were specific, because no signal was obtained in the absence of the primary HEX antibody and in cells or specimens negative for HEX expression (HeLa cells and medullary carcinomas; data not shown). Nuclear localization was observed in 30% of TFCs. Very similar values were detected in thyroid adenomas. In sharp contrast, both differentiated and undifferentiated thyroid carcinomas showed only cytoplasmic localization of HEX protein (Fig. 3B). These results are consistent with data obtained by RT-PCR, indicating that cell transformation, in most cases, does not abolish HEX expression. In addition, immunohistochemistry data reveal that the HEX cell localization is greatly modified in cancer cells. In particular, HEX nuclear localization appears to be abolished by cell transformation. Some evidence indicates that the difference in HEX localization between thyroid adenomas and carcinomas is not due to technical reasons. First, several of the neoplasms analyzed in this study have also been investigated for Ref-1 expression, which, in thyroid cells, is present both in the nucleus and in the cytoplasm (42). In all of these cases, Ref-1 immunostaining was detectable. Second, the normal tissue surrounding tumoral cells, when present, always showed normal HEX expression in terms of both positivity and subcellular localization.
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. In all cell lines, HEX was detected only in cytoplasm. Identical results were obtained when cytoplasmic and nuclear extracts of BCPAP, WRO, FRO, and ARO cells were fractionated and subjected to Western blot (Fig. 4B). In all cell lines, HEX was present in cytoplasmic, but not in nuclear extracts. By using the same procedure, normal rat thyroid FRTL-5 cells showed both nuclear and cytoplasmic localization (data not shown).
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To test whether the disappearance of HEX nuclear localization correlates with some other features of the thyroid malignancies investigated, several clinical, pathological, and molecular features (previously detected) of these tumors were analyzed. Among the papillary carcinomas, dimensions ranged from 1–2.5 cm, and 3 of 20 showed lymph node metastases. From the histological point of view, 2 of 20 papillary carcinomas belong to the follicular variant. Albeit at variable levels, all tested papillary carcinomas express Tg and TPO. Five of 20 do not express NIS. TTF-1 expression was detectable in all cases, whereas PAX8 expression was abolished in 6 of 20 samples. Also, follicular carcinomas were heterogeneous in terms of clinical, pathological, and molecular features. A major difference with papillary carcinomas was PAX8 expression, which can be detected in only 3 of 10 follicular carcinomas. In anaplastic carcinomas, Tg and TPO mRNAs were barely detectable. All of them were negative for TTF-1 and PAX8 expression. The only undifferentiated cancer negative for HEX expression did not show significant differences to other undifferentiated cancers analyzed in this study. Altogether, these data indicate that the disappearance of nuclear localization observed in thyroid malignancies is not associated to any of the clinical, pathological, and molecular features here evaluated.
Discussion
Alteration in the control of gene expression is considered as a key event in neoplastic transformation, responsible for the loss of the physiological regulation of both cell proliferation and differentiation occurring in cancer cells. Because gene expression is mostly under the control of tissue-specific transcription factors, investigation of the functions and expression of these proteins may allow a better characterization of the molecular mechanisms underlying changes of a given cell type during neoplastic transformation. Moreover, these studies could provide new information for a neoplasm classification founded on molecular-based criteria. In this view, thyroid tumors represent an excellent model of investigation. In fact, at least four tissue-specific transcription factors (TTF-1, TTF-2, PAX8, and HEX) have been characterized (11, 29). Moreover, different histotypes of tumors, representing different stages of the tumoral progression (from the well differentiated to the completely undifferentiated ones) may be examined, and several cell lines derived from normal and tumoral tissues or expressing different oncogenes are also available as in vitro models (43).
Changes in the expression of the thyroid-specific transcription factors TTF-1 and PAX8 have been demonstrated already in human thyroid tumors, contributing to the characterization of these neoplasms (14). The interest in studying HEX gene expression in human thyroid tumors stems from observations indicating a dual role played by HEX in thyroid cells. In fact, during development, HEX is required to the progression of endodermal cell precursors to the terminal differentiated state (28); in the adult, it contributes to the regulation of tissue-specific gene expression (29).
TTF-1 and PAX8 on one hand, and HEX on the other, are expressed in a different manner during thyroid and primitive pharynx development. In fact, during mouse development, TTF-1 and PAX8 begin to be expressed in epithelial cells of primitive pharynx at 9.5 d post coitum, just when the thyroid bud is appearing (9, 44). In contrast, HEX expression begins much earlier, in primitive endoderm of implanting blastocyst, and only at later stages it is restricted to endodermal cells fated to generate the thyroid gland (28). These differences suggest that regulatory mechanisms controlling HEX expression could be different from those controlling either TTF-1 or PAX8. This hypothesis is corroborated by our present data on undifferentiated thyroid carcinomas. In fact, although TTF-1 and PAX8 are never expressed in undifferentiated carcinomas (14, 15, 16), HEX gene is still expressed in the majority of these cancers. The lack of expression of the HEX transcript in some undifferentiated carcinomas (one of five, in our series) may be expected when considering the wide genetic abnormalities commonly observed in this type of tumor. Identical results have been obtained by using 30- and 40-cycle amplifications. It could not be excluded that, by reducing the number of PCR cycles, some samples would show no HEX expression. In that case, however, negative samples would be due to reduction, but not absence, of HEX expression. A quantitative analysis of HEX mRNA expression is beyond the aim of the present study.
Our previous data showed the absence of HEX gene expression in some rat thyroid tumoral cell lines presenting a highly malignant phenotype (29). In contrast with such in vitro data, our present findings show that HEX expression is maintained in all differentiated thyroid tumors investigated, both benign and malignant, and, differently from TTF-1 and PAX8, also in the majority of undifferentiated carcinomas. More interestingly, we found that only in the malignant tumors, and not in thyroid adenomas, HEX expression is restricted to the cytoplasm compartment. The abolition of HEX nuclear localization in thyroid malignancies should not be a simple epiphenomenon of the malignant transformation. In fact, the leukemia K562 and NB4 cell lines still retain HEX nuclear localization (35). A major question raised by our data is related to whether HEX functions in the cytoplasm. Two alternative hypotheses can be delineated. The first is that HEX has cytoplasmic functions, which are unaffected during neoplastic transformation. Which functions can HEX play outside the nucleus? In the case of another homeodomain-containing protein, Engrailed, it has demonstrated an association with sphingolipid-cholesterol microdomains (45), which are specialized in signal transduction and cycle between the plasma membrane and different luminal compartments (46). Moreover, Engrailed can be secreted by COS cells and internalized by neurons (47). A second possibility is that HEX compartmentalization is only a mechanism to regulate its nuclear functions. Indeed, control of subcellular localization provides a way to regulate the function of other transcription factors. For example, NF-
B is tethered in the cytoplasm by association with its partner IB, which masks the NF-B nuclear localization signal. The NF-B nuclear entry may be determined by several cellular stimuli, which activate IB degradation (48).
It has been well established that many homeobox genes regulating embryogenesis can, if deregulated, also contribute to tumorigenesis (6). In particular, several studies have proven a critical involvement of homeobox genes in tumorigenesis of the hemopoietic cell line (reviewed in Ref. 49). Among them, HEX mRNA was detected in leukemic cells, even if its expression is lost upon induction of differentiation into monocytes, macrophages, and megakaryocytes (27). Few data have been published so far on the behavior of HEX during neoplastic transformation of epithelial tumors; Tanaka et al. showed that HEX expression is detectable in highly differentiated hepatoma cells but is abolished in poorly differentiated hepatoma cell lines (40). To our knowledge, this is the first study addressing the involvement of HEX protein in human tumoral epithelial tissues. Our data demonstrate that the alteration of the subcellular localization of this tissue-specific transcription factor is a peculiar feature of malignant vs. benign thyroid tumors, independent from the histotype or the degree of aggressiveness of the neoplasia. These data deserve to be confirmed prospectively to evaluate their role in improving the diagnostic accuracy of conventional cytology of thyroid nodules.
Acknowledgments
Footnotes
This work is funded by grants from the Consiglio Nazionale delle Ricerche (Target project on Biotechnology), and Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST) (to G.D.), by Grant Progetto Giovani Ricercatori 2000 from the University of Trieste (to G.T.), by a grant from the University of Trieste (to G.M.), and by grants from Associazione Italiana Ricerca Cancro and MURST (to S.F.).
Abbreviations: NIS, Sodium/iodide symporter; TFC, thyroid follicular cell; TPO, thyroperoxidase.
Received July 30, 2001.
Accepted December 10, 2001.
References
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