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Istituto Nazionale dei Tumori di Napoli, Fondazione Senatore Pascale, 80131 Naples, Italy [G. C., M. C. D. B., F. P., F. d. N., S. L.]; Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510 [G. T.]; Dipartimento di Biologia Cellulare e Molecolare, Centro di Endocrinologia ed Oncologia Sperimentale del C.N.R., Facoltà di Medicina e Chirurgia, Università degli Studi di Napoli, Naples, Italy [M. F., S. B., M. S.]; Istituto Internazionale di Genetica e Biofisica, Consiglio Nazionale delle Ricerche, 12-80125 Naples, Italy [P. V.]; and Dipartimento di Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia di Catanzaro, Università degli Studi di Catanzaro, Catanzaro, Italy [A. F.]
ABSTRACT
fra-1 gene overexpression has been shown to represent a general event in thyroid cell transformation in vitro and in vivo. Moreover, inhibition of FRA-1 protein synthesis by stable transfection with a fra-1 antisense construct significantly reduces the malignant phenotype of the transformed thyroid cells, indicating a pivotal role of the fra-1 gene product in the process of cellular transformation. In the attempt to define the potential use of FRA-1 protein detection in the diagnosis of thyroid diseases, we analyzed Fra-1 expression by a combination of immunohistochemistry and reverse transcription-PCR (RT-PCR) assay in 174 samples of thyroid nodules (22 nodular hyperplasias, 102 follicular adenomas, 34 papillary carcinomas, 12 follicular carcinomas, and 4 anaplastic carcinomas) representative of the spectrum of thyroid tumor pathology. FRA-1 protein was abundant in all of the carcinoma samples (50/50, 100%), with an intense staining in the nucleus and the cytoplasm. Positive staining was also found in most of the adenomas (90 of 102; 88%), but in this case, the staining was restricted to the nucleus. Similar results were obtained from the analysis of thyroid goiters; however, the number of positive cases is lower than adenomas (8 of 22; 36%); moreover, the staining was not observed in all of the cells. Conversely, no FRA-1 protein was detectable in 12 normal thyroid tissue samples used as controls. RT-PCR analysis confirmed a higher fra-1 expression in papillary and follicular carcinomas compared with goiters and adenomas. fra-1 expression was also analyzed on 10 fine needle aspiration biopsy (FNAB) samples by RT-PCR. fra-1-specific mRNA was detected in seven of the eight FNABs corresponding to thyroid nodules that were eventually diagnosed as adenomas (three of four) and carcinomas (four of four) after surgery. Conversely, no fra-1 gene expression was observed in two FNABs derived from normal thyroid. Further studies are required before suggesting FRA-1 protein detection as a useful tool for the diagnosis of hyperplastic and neoplastic disorders of the thyroid gland.
INTRODUCTION
The FRA-1 protein is a member of the AP-1 complex that is formed by the three Jun family members (c-Jun, JunB, and JunD) and four Fos family members (c-Fos, FosB, Fra-1, and Fra-2; Refs. 1, 2, 3, 4, 5 ). Each of these Fos-related proteins is a transcription factor that can dimerize with Jun family proteins through a leucine zipper domain and bind through a conserved basic region to very similar or identical DNA motifs. The DNA element recognized by all of these heterodimers is an AP-1 site with core sequence TGACTCA (6, 7, 8) . However, the Fos proteins cannot dimerize among themselves and have no intrinsic specific DNA binding activity (9) .
Modification in AP-1 activity has been often correlated with cell transformation (8) . In fact, c-jun knock-out mice-derived cells are resistant to ras-mediated transformation (10) . Moreover, fra-2-c-jun heterodimers play a crucial role in chicken fibroblast transformation (11) . Mouse JunD antagonizes transformation by ras (12) , and c-fos expression is required for malignant progression of skin tumors (13) . We have demonstrated recently that thyroid neoplastic transformation induced by the v-ras-Ki and v-mos oncogenes is associated with a drastic increase of AP-1 activity, which reflects multiple compositional changes (14) . The main effect is represented by a dramatic fra-1 gene induction. Although no specific biological effect was observed after fra-1 gene overexpression, the inhibition of the FRA-1 protein synthesis by stable transfection with a fra-1 antisense construct significantly reduces the malignant phenotype of the transformed thyroid cells, indicating a pivotal role of the fra-1 gene product in the process of cellular transformation (14) . Our recent results showing that the induction of the fra-1 gene is present even in cells transformed with several oncogenes (E1A gene of adenovirus, RET/PTC, v-raf, v-abl, Middle T of polyoma, v-src, and others) and in human thyroid carcinoma cell lines of different histotype indicate that the fra-1 gene expression induction is a general event in the process of thyroid carcinogenesis (15) . Moreover, in rat fibroblasts fra-1 exhibits oncogenic potential because its overexpression has been shown to be unable to induce morphological transformation, but capable to stimulate anchorage-independent growth (16) .
fra-1 gene expression is subject to positive control by AP-1 activity (15 , 17) ; however, AP-1-induced expression of fra-1 depends on regulatory sequences located not only in the promoter region but also in the first intron. Because fra-1 gene expression is prevented by the block of the HMGI-C protein synthesis, the possibility that fra-1 gene expression is directly or indirectly regulated by the expression of the HMGI proteins should also be taken into account. The presence of five consensus binding sites for members of the HMG-box family (18) in the fra-1 promoter is consistent with this hypothesis.
Thyroid nodules are frequently found in the general population. The large majority of them are benign, and 5–10% are eventually diagnosed as carcinomas only after surgical removal of the lesion. Evaluation of the potential of molecular markers in the diagnosis of thyroid nodules is therefore justified. In this context, we analyzed FRA-1 expression by immunohistochemistry and RT-PCR3 in nodular lesions of the thyroid gland to assess its possible role as a diagnostic tool.
The results presented here show the expression of the FRA-1 protein in all of the thyroid carcinomas analyzed. Eighty-eight % of thyroid adenomas and 36% of goiters were positive, but in these cases the staining was restricted to the nucleus. RT-PCR analysis confirmed a lower fra-1 gene expression in adenomas. Conversely, FRA-1 protein was not detected at all in normal thyroid tissue.
MATERIALS AND METHODS
Tissue Samples.
Tissue sections for immunohistochemistry were obtained from 172
routinely processed, paraffin-embedded samples of thyroid nodules
resected at Yale New Haven Hospital between 1985 and 1990 and at the
Istituto dei Tumori di Napoli between 1989 and 1995. The thyroid
nodular lesions were diagnosed as nodular hyperplasia (22 cases),
follicular adenoma (102 cases), papillary carcinoma (34 cases),
follicular carcinoma (12 cases), and anaplastic carcinoma (4 cases).
Twelve samples of histologically normal thyroid were also included as
controls. None of the adenomas analyzed may be considered as atypical.
RNA for RT-PCR was extracted from paraffin blocks on a subset of these
cases to include 4 normal thyroids, 8 nodular hyperplasias, 10
adenomas, and 15 carcinomas. Eight FNAB samples were obtained at the
Istituto dei Tumori di Napoli for RNA extraction and RT-PCR. The final
diagnoses were follicular adenoma (4 cases), papillary carcinoma (2
cases), and follicular carcinoma (2 cases).
Thyroid Cell Lines.
The FRO cell line was derived from a human anaplastic thyroid carcinoma
(19
, 20)
. They were grown in DMEM plus 10% FCS. HTC-2
cells were established and cultured as described (21)
.
Immunohistochemistry.
For the immunohistochemical studies of paraffin-embedded samples,
5–6-µm-thick paraffin sections were deparaffinized and then placed
in a solution of absolute methanol and 0.3% hydrogen peroxide for 30
min and then washed in PBS before immunoperoxidase staining. Slides
were then incubated overnight at 4°C in a humidified chamber with the
primary antibodies diluted 1:100 in PBS and subsequently incubated,
first with biotinylated goat antirabbit IgG for 20 min (Vectostain ABC
kits; Vector Laboratories) and then with premixed reagent ABC (Vector)
for 20 min. The immunostaining was performed by incubating slides in
diaminobenzidine (Dako) solution containing 0.06 mM
diaminobenzidine and 2 mM hydrogen peroxide in 0.05% PBS
(pH 7.6) for 5 min, and after chromogen development, slides were
washed, 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 rabbit polyclonal raised against the epitope corresponding to amino acids 3–22 mapping at the NH2 terminus of FRA-1 of human origin. They are specific for FRA-1 and non-cross-reactive with the other members of the fos family. The immunostained samples were blindly read by two independent individuals (G. C. and G. T.).
RT-PCR Analysis of fra-1 Gene Expression.
RNA was extracted from paraffin-embedded blocks on 37 cases that were
analyzed in parallel for FRA-1 expression by immunohistochemistry. RNA
extraction was performed as described (22)
. Briefly,
single 6–8-mm tissue sections, cut from paraffin blocks, were stirred
for 20 min in 1.5-ml tubes with 1 ml of xylene. After centrifugation,
the pellet was washed with 0.5 ml of ethanol and air-dried. The dried
pellet was resuspended in 200 ml of 6 mg/ml proteinase K (Sigma
Chemical Co., St. Louis, MO), 1 M guanidinium thiocyanate,
25 mM 2-mercaptoethanol, 0.5% Sarkosyl, and 20
mM Tris-HCl (pH 7.5) and incubated at 37°C for 18 h.
RNA was then extracted with phenol and precipitated with ethanol
following a standard procedure (23)
. Fine needle
aspiration samples were washed twice with 1x PBS and then processed
for RNA extraction following the same procedure. One-fifth of RNA of
total RNA, digested with DNase, was reverse transcribed using random
exanucleotides as primers (100 mM) and 12 units of AMV
reverse transcriptase (Life Technologies, Inc.), and subsequent PCR
amplification was performed as reported previously (24)
.
Two hundred ng of cDNA were amplified in a 25-µl reaction mixture
containing Taq DNA in polymerase buffer, 0.2 mM
deoxynucleotide triphosphates, 1.5 mM
MgCl2, 0.4 mM of each primer, and 1
unit of Taq DNA polymerase (Perkin-Elmer). The PCR amplification was
performed for 30 cycles (94°C for 30 s, 55°C for 2 min, and
72°C for 2 min). The specific primers for fra-1 were:
forward, 5'-GTCATTGCTAGGATACCAAAC-3'); and reverse,
5'-CACTGTCCAGCAAGGGTCTGT-3', corresponding to the nucleotides 136–156
and 335–315, respectively (4)
. The amplified products
were separated by 1.5% agarose electrophoresis gel and hybridized with
a fra-1 probe. Amplification of contaminating genomic DNA
was excluded by control experiments in absence of reverse
transcriptase. In addition, a set of primers specific for the
constitutively expressed enzyme GAPDH was added to each reaction after
20 cycles of PCR to serve as internal control for the amount of
cDNA tested. The GAPDH-specific primers were: forward,
5'-ACATGTTCCAATATGATTCC-3' corresponding to the nucleotides 194–214;
and reverse, 5'-TGGACTCCACGACGTACTCAG-3' corresponding to the 336–356
nucleotides. The product of the reaction was analyzed on a 2%
agarose gel and then transferred by electroblotting to GeneScreen plus
nylon membrane (DuPont, Boston, MA). DNA was fixed to the membranes by
air drying and UV cross-linking, and then membranes were hybridized
with a GAPDH probe. A PhosphorImager screen was briefly exposed to the
membranes, and the screen was then scanned on a Molecular Dynamics
PhosphorImager. The images recorded by the PhosphorImager were analyzed
by volume integration with the ImageQuant software. The relative level
of fra-1 expression was assessed by comparison with the level of GAPDH
in the same sample.
RESULTS
Immunohistochemical Analysis of fra-1 Gene
Expression.
Detection of FRA-1 protein by immunohistochemical analysis allows a
rapid and sensitive screening of thyroid pathological tissues and is
amenable to regular use as a routine diagnostic test. This technique
was therefore chosen for FRA-1 protein analysis using antibodies
raised, in our laboratory, against a FRA-1-specific peptide. To find
the best experimental conditions, FRO cells and tumors induced by
injecting the FRO cell line into athymic mice were used as positive
controls (20)
. The FRO cell line was chosen because of its
high expression of the Fra-1 protein by Western blot analysis
(15)
. No staining was observed with human thyroid cells
(HTC-2 cells; Fig. 1
A; Ref.
21
), whereas a positive nuclear staining was obtained with
FRO cells (Fig. 1
B) and FRO-induced tumors (Fig. 1
C).
|
. No staining
was observed in normal thyroid tissue (Fig. 2)
. Conversely, strong immunoreactivity
was detected in all of the thyroid carcinomas analyzed (Fig. 2)
. The
positivity appears in the nucleus, but it is also present, although
slightly weaker, in the cytoplasm. This result is consistent with
previous published data that showed FRA-1 protein localization in the
nucleus and the cytoplasm of the COS cells (25)
.
Eighty-eight % of follicular adenomas and 36% of the goiters were
positive for FRA-1. However, in these cases, the immunoreactivity was
always restricted to the nuclei. Moreover, in goiters the nuclear
staining was not observed in all of the cells. In Fig. 2
, we show some
examples of the immunohistochemical assay. No positive signal was
detected in normal thyroid (Fig. 2
A) and in one goiter (Fig. 2
F), whereas an intense nuclear and a weaker cytoplasmic
staining was clearly observed in a papillary carcinoma (Fig. 2
C) and in a follicular carcinoma (Fig. 2
E). Fig. 2
, G and H, shows the immunostaining of one
positive goiter and one positive adenoma, respectively; the
immunoreactivity is present only in the nuclei. For each case, sections
were stained without the primary antibody, and in all cases, these
controls were negative (Fig. 2
D). No staining was also
observed when the neoplastic tissues were analyzed after the
FRA-1-specific antibodies had been preincubated with the FRA-1 control
peptide (Fig. 2
B). In Fig. 3
,
we show that fra-1 detection was specific for the neoplastic cells,
because no staining was observed in the normal follicles present in the
same field.
|
|
|
. Fig. 4
shows a representative RT-PCR assay. Normal thyroid was negative for
the fra-1 gene expression (Lane 1). Three
adenomas (Lanes 3, 4, and 5) of four
(Lane 2) were positive. Conversely, all of the
malignant tumors showed a significant fra-1 expression (from
Lanes 6 to 15). In thyroid malignant neoplasias,
the expression was higher (at least 4–5-fold) in comparison with
benign lesions. The fra-1 gene expression in goiters was
slightly lower than that observed in adenoma samples (data not shown).
Therefore, these data support the immunohistochemical findings.
|
and Fig. 5
, are consistent with the
immunohistochemical findings. The fine needle biopsies from patients
carrying a carcinoma showed expression of the fra-1 gene
(Lanes 5, 6, 7, and 8),
whereas the fine needle biopsy from normal thyroid (Lanes 1
and 9) was negative. Two adenoma samples were positive for
the fra-1 gene expression (Lanes 3 and
4), and one scored negative (Lane 2).
No amplification product was achieved when a carcinoma RNA sample was
analyzed in absence of reverse transcriptase (data not shown).
|
|
Thyroid tumors include a wide spectrum of lesions with different phenotypic characteristics and biological behavior: benign adenomas, differentiated carcinomas, and anaplastic carcinomas (26) . We have demonstrated previously that the chromatinic proteins belonging to the group of the high mobility proteins are expressed at high levels in neoplastically transformed thyroid cells in culture and in human carcinomas, whereas they were not detected in normal thyroid and goiters and are present in 20% of adenomas (19 , 20) . Recently, the analysis of the AP-1 complex in thyroid cells transformed by MPSV and KiMSV revealed the induction of fra-1 in transformed thyroid cells. This induction was abolished by blocking the HMGI protein synthesis, suggesting a regulation of the fra-1 gene by the HMGI proteins (14) . Rat and human thyroid carcinoma cell lines showed expression of the fra-1 gene, which was absent in normal rat thyroid cells and in human thyroid tissue (15) . In addition, the inhibition of FRA-1 protein synthesis by stable transfection with a fra-1 antisense RNA vector significantly reduced the malignant phenotype of transformed thyroid cells, indicating a pivotal role for the fra-1 gene product in the process of cellular transformation (14) . Recent data showing immunohistochemical evidence of FRA-1 protein expression in a variety of breast, ovarian, and skin carcinomas4 but not in the corresponding normal tissues suggest that fra-1 induction is a rather common event in the process of epithelial cell transformation and that FRA-1 protein detection might, therefore, represent a useful tool for the diagnosis of neoplastic and/or proliferative processes.
The aim of the present study was to investigate whether the
detection of the FRA-1 protein in the thyroid neoplastic samples could
have been of some help in the diagnosis. In the present study, we
analyzed 50 thyroid carcinomas (34 papillary, 12 follicular, and 4
anaplastic), 102 adenomas, 22 goiters, and 12 thyroid normal tissue
samples for the presence of the FRA-1 protein by immunohistochemical
analysis and RT-PCR. The presence of the FRA-1 protein was not detected
in normal thyroid; it did not, however, appear restricted to thyroid
malignant neoplasias. In fact, 36% of the hyperplastic nodules
analyzed were positive immunohistochemically for the presence of
fra-1 gene product. However, the modality of staining was
different from goiters and adenomas in comparison with the malignant
neoplasias, because nuclear and cytoplasmic staining was observed in
papillary and follicular carcinomas, whereas the immunoreactivity was
restricted to nuclei in positive adenomas and goiters. These results
were confirmed by RT-PCR analysis. In fact, they show a higher
fra-1 gene expression in carcinomas, compared with adenomas
and goiters. Analysis of the FNAB confirms the expression in the case
of benign and malignant tumors but not in normal thyroid samples.
Therefore, the presence of the FRA-1 protein, which is not detectable
at all in normal human thyroid, surely indicates a thyroid cell
proliferation, and then it is nevertheless a marker for proliferative
processes in thyroid (both hyperplastic and neoplastic). Moreover, the
kind of immunoreactivity may allow a discrimination between malignant
and benign neoplasias, because the positivity is restricted to the
nuclei in adenomas. Finally, FRA-1 protein detection may
represent, in association with expression analysis of other genes, such
as HMGI(Y) and/or ß-10 thymosin
(27)
, a useful adjuvant for the diagnosis of thyroid
lesions. In fact, HMGI(Y) and FRA-1 proteins are absent in normal
thyroid and are induced in all carcinomas. However, a differential
expression was observed in thyroid adenomas; FRA-1 protein is present
in most adenomas (88% of cases), whereas HMGI(Y) protein is detectable
in only 
20% of follicular adenomas (28)
. Therefore,
the detection in thyroid adenomas of FRA-1, but not HMGI (Y), might
exclude the diagnosis of follicular carcinomas. Conversely, this
diagnosis might be taken in consideration when both these proteins are
expressed, especially if FRA-1 protein is detected in the cytoplasm as
well. At the same time, the negativity of a thyroid sample for both the
genes might exclude the diagnosis of a hyperproliferative process.
However, to assess the potential use of FRA-1 detection in the
diagnosis of thyroid neoplasias, a higher number of normal thyroid,
follicular, and anaplastic carcinoma samples must be analyzed.
The expression of the fra-1 gene in human adenomas is consistent with the results regarding in vitro thyroid cell transformation. In fact, we showed that fra-1 was expressed in all of the rat thyroid transformed cell lines, even in those that did not show a fully malignant phenotype, such as PC E1A, PC PTC, and PC Harvey (15) .
In conclusion, fra-1 gene activation appears to be an early event in the process of thyroid carcinogenesis, and its detection may represent, together with the analysis of other markers, a useful tool in the diagnosis of human thyroid neoplastic diseases.
ACKNOWLEDGMENTS
We thank Annamaria De Bernardo for editing the manuscript.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the "Progetto
Biotecnologie Mediche ed Agroalimentari" del Parco Scientifico et
Tecnologico dell’Area Metropolitana di Napoli. 
2 To whom requests for reprints should be
addressed, at Dipartimento di Biologia e Patologia Cellulare e
Molecolare, Facoltà di Medicina e Chirurgia di Napoli,
Università degli Studi di Napoli "Federico II," via Pansini
5, 80131 Naples, Italy. Phone: 39-081-7463056; Fax: 39-081-7463037 or
-7701016; E-mail: afusco{at}napoli.com
3 The abbreviations used are: RT-PCR, reverse
transcription-PCR; FNAB, fine needle aspiration biopsy; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; HMGI, high-mobility group
I.
Received 4/13/00; revised 6/22/00; accepted 7/ 6/00.
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