Full text of DOI:10.1089/10507250150500612

THYROID
Volume 11, Number 1, 2001
Mary Ann Liebert, Inc.
Overexpression of Epidermal Growth Factor and
Epidermal Growth Factor-Receptor mRNAs in
Dyshormonogenetic Goiters
Filippo Pedrinola, Ileana Rubio, Cecilia L.S. Santos, and Geraldo Medeiros-Neto
Thyroid malignancy has been induced by long-term endogenous thyrotropin (TSH) stimulation in experimen-
tal animals, leading to local and distant metastasis. It has been postulated that constant and prolonged en-
dogenous TSH stimulation in dyshormonogenetic thyroid tissues could result in thyroid neoplasia. The possi-
ble role of growth factors and oncogenes in goitrogenesis and favoring neoplasia has also been mentioned.
Overexpression of certain growth factors and/or their receptors, and of oncogenes implicated in growth pro-
motion may play a significant role in the relatively frequent finding of thyroid malignancy in congenital goi-
ters. In this study the expression of epidermal growth factor (EGF), epidermal growth factor receptor (EGF-R),
transforming growth factor-b (TGF-b), c-myc, and p53 mRNAs was determined in 14 thyroid tissue samples:
6 from patients with thyroid peroxidase (TPO) gene mutations, 4 with thyroglobulin (Tg) gene defects and 4
normal thyroid tissues. EGF mRNA overexpression was seen in 7 of 10 dyshormonogenetic tissues (3.5 to 12.0
arbitrary optical densitometry units [AODU]) and considered significantly higher ( , 0.01) when compared
p
to normal thyroid tissues (0.25 to 0.32 AODU). Moreover, overexpression of EGF-R mRNA was present in 6 of
10 dyshormonogenetic tissues (2.23 to 13.03 AODU) and considered significantly higher ( , 0.01) when com-
p
pared to normal thyroid tissues (0.42 to 0.65 AODU). There was no difference in c-myc, p53, and TGF-b
mRNAs expression between dyshormonogenetic and normal tissues. The overexpression of EGF and EGF-R
mRNAs found in dyshormonogenetic tissues may suggest that this growth factor may play a role in cellular
proliferation and contribute to goiter formation.
logical studies of congenital goiters (5–11), as compared with
an incidence of 3%–6% in multinodular sporadic goiter (12).
Introduction
ONGENITAL GOITER, frequently associated with hypothy- Most of these malignant tissues have been found in long-
roidism, is a group of genetically induced thyroid con- standing congenital goiters in patients who refused surgery.
C
ditions that show biochemical abnormalities in one or more The real incidence of malignancy in congenital goiter pa-
steps of thyroid hormone synthesis. Elevated serum thy- tients is difficult to ascertain because most individuals are
rotropin (TSH) levels are commonly found in these condi- submitted to subtotal thyroidectomy relatively early in post-
tions and often can lead to an extremely high degree of hy- natal life, the resulting surgical hypothyroidism being com-
perplasia (1–4). As postulated previously, patients with pensated by levothyroxine (LT4) substitutive therapy. Nev-
congenital goiter offer an unique opportunity to examine po- ertheless, thyroid remnant growth after surgery is a common
tential human tumorigenesis as the endogenous TSH stimu- finding in these patients, suggesting the influence of thyroid
lation starts at birth or even during fetal life (5).
growth factors aside from the presence of endogenous TSH
It is reasonable to consider the possibility that prolonged usually suppressed by the LT4 therapy (5). Abnormal ex-
and intense endogenous stimulation by TSH could lead to pression of growth factors, their respective growth factor re-
increased thyroid tumorigenesis (4,5). The incidence of thy- ceptors, and oncogenes may be involved in the goitrogene-
roid cancer in congenital goiter has been estimated to be ap- sis of dyshormonogenetic thyroid tissue.
proximately 18% based on 19 reported cases in 109 patho-
Several growth factors or local paracrine synthesized sub-
Laboratório Molecular de Tireoide (LIM-25), Thyroid Unit, Division of Endocrinology, University of São Paulo Medical School, São
Paulo, Brazil.
15

16
PEDRINOLA ET AL.
stances have mitogenic action over thyroid cells
normal tissues, six goitrous tissues from patients with thy-
in vitro
(13–15). Among these are the epidermal growth factor (EGF) roperoxidase (TPO) gene mutations, and four goitrous tis-
and its receptor (EGF-R). Recently the vascular endothelial sues from patients with thyroglobulin (Tg) gene defects.
growth factor (VEGF) has also been implicated in thyroid tu-
Total RNA was extracted according to the procedure of
mor growth and differentiation (16). At least one local fac- Chomczynski and Sacchi (28). The integrity of each sample
tor inhibits thyroid cells growth: the transforming growth was examined by electrophoresis, through a 1% agarose gel
factor- (TGF- ) (13). These local factors may also act in en- and ethidium bromide staining. The quantity of total RNA
b
b
dothelial cells and in thyroid fibroblasts mediating cross-talk was assessed by spectrophotometric absorbance at 260 nm
that will permit a balanced growth of the thyroid gland. (Beckman DU-64, Beckman, Germany). Total RNA prepara-
TSH is the classic stimulator of thyroid cellular function. tions were divided into aliquots of 4 g per tube with 1 U/ L
m
m
TSH acts via the adenylate cyclase system and the phospholi- RNAsin (Promega Corp., Madison, WI) and stored at 80°C.
2
pase C cascade and, experimentally, it is widely accepted that
TSH stimulates growth and cell differentiation (13).
Semiquantitative RT-PCR
EGF acts via its receptor (EGF-R) via the tyrosine-kinase sys-
tem and is an important growth factor in certain types of thy-
roid cancer (17–22). As opposed to TSH, EGF stimulates thy-
roid cellular growth but inhibits cell differentiation (19–22).
p53 is a tumor suppressor gene that is activated in re-
sponse to DNA damage and/or other cellular stressors ar-
rests cells in the G1/S boundary, and can trigger apoptosis
when serious DNA damage occurs (23,24). Inactivation of
p53 has been implicated in thyroid malignant transforma-
Total RNA from thyroid biopsy specimens were first di-
gested with DNAse I before being used as template for first-
strand cDNA synthesis with oligo dT and Super Script TM
Reverse Transcriptase (Gibco BRL, Gaithersburg, MD). Poly-
merase chain reactions (PCRs) were performed with the in-
tron spanning primers depicted in Table 1 (all purchased
from Stratagene, La Jola, CA, except for GAPDH-340, which
was kindly provided by Dr. Peter Kopp.
In all PCR reactions, a negative control without reverse tran-
scriptase was included. As an internal control for cDNA tem-
plate loading, we performed PCR analysis for constitutively
expressed GAPDH (glyceraldehyde-3-phosphate dehydroge-
nase) mRNA. Serial dilutions of cDNA were amplified in a
tion (24). The c-
myc
proto-oncogene codes for a nuclear pro-
tein that is overexpressed in some thyroid cancers and acts
as a transcription factor (26). c- mRNA expression shows
myc
positive correlation with thyroid cancer aggressiveness and
differentiation (26,27).
In this article we examine the expression of EGF, EGF-R,
two-step PCR using
polymerase (Gibco BRL). In the first
Taq
step, only the primers of the investigated mRNA were used,
while in the second step the primers of the internal control
gene (GAPDH) were added. Annealing temperature and
number of cycles were established for each gene (Table 2).
Amplification products were examined by 1.5% agarose
gel electrophoresis and ethidium bromide staining (Fig. 1).
The size of DNA fragments was determined by comparison
with a 100-bp or 1-kb ladder molecular weight markers
(Gibco BRL) and densitometries of the amplification prod-
ucts were performed using the “One Scan” Stratagene pro-
gram (Stratagene). Relative concentration of mRNA was ob-
tained by dividing the densitometric value of each analyzed
factor by the densitometric values of internal control
TGF- , c-myc, and p53 mRNAs in 10 dyshormonogenetic
b
thyroid tissues specimens and compared the results with
four normal thyroid tissues. We found evidence for EGF and
EGF-R mRNAs overexpression in dyshormonogenetic tis-
sues, suggesting a possible role of these factors in abnormal
cellular growth.
Materials and Methods
Isolation of RNA from thyroid biopsy specimens
Fourteen thyroid biopsy specimens were obtained at
surgery and immediately immersed in liquid nitrogen: four
TABLE 1. PRIMERS USED IN PCR AMPLIFICATIONS
Product
size (bp)
Gene
Oligonucleotide sequences
F: 5 -CAGGTAATGGAGCGAAGCTTTCAT-3
Nucleotide no.
EGF
3910–4013
4534–4511
933–947
1469–1455
238–256
583–566
517–539
947–928
604–627
913–890
40–57
624
540
345
431
310
340
600
9
9
9
R: 5 -GAGTTAAATGCCTACACTGTATCT-3
9
EGF-R
F: 5 -GGTCTGCCGCAAATT-3
9 9
R: 5 -GCCGCGTATGATTTC-3
9
9
c-
myc
F: 5 -CCAGCCAGCGACTCTGAGG-3
9 9
R: 5 -CCAAGACGTTGTGTGTTC-3
9
9
P53
F: 5 -CCTGCCCTCAACAAGATGTTTTG-3
9 9
R: 5 -TCAAAGCTGTTCCGTCCCAG-3
9
9
TGF-
F: 5 -TCCAAAGATTTAACATCTCCAACC-3
9 9
b
R: 5 -TCCCACTGTTTTTTTTCCTAGTGG-3
9
9
GAPDH-340
GAPDH-600
F: 5 -GAGCCACATCGCTCAGAC-3
9 9
R: 5 -TTCTCCATGGTGGTGAAG-3
380–363
212–236
811–787
9
9
F: 5 -CCACCCATGGCAAATTCCATGGCA-3
9
9
9
R: 5 -TCTAGACGGCAGGTCAGGTCCACC-3
9
PCR, polymerase chain reaction; EGF, epidermal growth factor; EGF-R, epidermal growth factor-receptor; TGF-b, transforming growth
factor-b; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

17
GROWTH FACTORS IN DYSHORMONOGENETIC GOITER
TABLE 2. APPROPRIATE CONDITIONS FOR THE SEMIQUANTITATIVE PCR FOR EACH STUDIED GENE
Genes
myc
Conditions
EGF
EGF-R
c-
p-53
TGF-b
Annealing temperature (°C)
One-step PCR
55
17
55
17
60
2
55
2
60
4
(number of cycles)
Two-step PCR
(number of cycles)
25
25
24
24
24
PCR, polymerase chain reaction; EGF, epidermal growth factor; EGF-R, epidermal growth factor-receptor; TGF-b, transforming growth
factor-b.
There was also increased EGF-R mRNA expression in 6 to
10 dyshormonogenetic tissues (2.23 to 13.03) as compared to
normal thyroid tissues (0.42 to 0.65 AODU) (Fig. 2). Spear-
man regression analysis was used in the statistical analysis
of the results. A significant positive correlation between EGF
and EGF-R mRNAs was found in normal and dyshormono-
GAPDH for each dilution. The final values are expressed by
arbitrary optical densitometry units (AODU). At least two
points within the exponential phase were selected and the
median obtained, representing quantification of expression
of each factor related to that of the internal control.
genetic tissues (Fig. 3). Expression of p53, c-
myc
and, TGF-
b
Patients
was not significantly different between normal and dyshor-
monogenetic tissues (data not shown).
All patients studied in this protocol gave their informed
consent and the project was approved by the Ethical Com-
mittee of the Hospital das Clinicas, University of Sao Paulo
Medical School.
The patients were identified as follows: subjects 1, 6, 13,
14, normal thyroid tissue (perinodular normal thyroid tissue
obtained at surgery); subjects 2, 3, 4, 5, 7, and 9, confirmed
TPO gene defects (mutations in the TPO gene); and subjects
8, 10, 11, and 12, confirmed Tg defective synthesis and se-
cretion (mutations in the Tg gene).
In spite of positive expression of TGF- mRNA in all an-
b
alyzed dyshormonogenetic tissues there was no statistically
significant difference between the dyshormonogenetic
goitrous tissues and the normal specimens.
Discussion
It is well accepted that proliferation of normal follicular
cells is regulated by stimulatory and inhibitory growth fac-
tors interacting with their respective membrane receptors
and transduction pathways. Any disorder affecting this com-
plex interaction network could lead to cellular growth ab-
normality.
Several reports have demonstrated that tumors arising
from a dyshormonogenetic goiter may have rapid growth
and behave aggressively with the occurrence of bone and
lung metastasis. This was particularly found in longstand-
ing congenital goiters (5–11).
Clinical, laboratory, and molecular details of studies con-
ducted in these patients can be obtained in previous publi-
cations by our group (1,2).
Expression of EGF and EGF-R messenger RNAs
There was a significant increase of EGF mRNA expression
in 7 of 10 dyshormonogenetic tissues (3.5 to 12.0 AODU) as
compared to normal thyroid tissues (0.25 to 0.32 AODU).
In this work, we have found significantly increased EGF
mRNA expression in 7 of 10 dyshormonogenetic tissues
when compared to normal thyroid specimens. From previ-
ously published work, it has been well documented (19–22)
that EGF stimulates thyroid cellular growth but has little role
in follicular cell differentiation. In porcine thyroid cells, TSH
through cyclic adenosine monophosphate (cAMP) induces
EGF-Rs making these cells more responsing to EGF. It is well
documented that EGF also induces proliferation of canine
thyroid cells (32,33). The growth of thyroid cells from other
species in culture (e.g., porcine, ovine, bovine, and human)
is also stimulated by EGF with the exception of the FRTL cell
line that lacks the EGF-R (20,34). Growth stimulation by EGF
is often weaker than that of TSH (20). However, the action
of EGF is accompanied by a general and reversible loss of
differentiation (20). This chain of events could take place in
dyshormonogenetic goiter tissues, providing an explanation
of how cellular growth induced by TSH may be potentiated
by locally expressed growth factors.
Representative result of quantitative reverse tran-
FIG. 1.
scriptase-polymerase chain reaction (RT-PCR). Ethidium
bromide staining of PCR products separated on a 1.5% poly-
acrylamide gel.
It is also well documented that EGF can act as a modula-
tor of thyroid growth and function
(19–21). Whereas
in vitro

18
PEDRINOLA ET AL.
volved in the regulation of the follicular cell growth in the
thyroid gland (20). Moreover, EGF has been reported to stim-
ulate the proliferation of follicular cells from various species
including humans (20,33). The presence of EGF-Rs has also
been detected in porcine and human thyroid cell membranes
(21). Papillary thyroid cancer tissues have increased expres-
sion of EGF-R and bind to more radiolabeled EGF than nor-
mal thyroid or adenomas (35). These findings indicate the
need to study the involvement of EGF-R and its ligands in
the control of proliferation of both normal and neoplastic
thyroid tissue.
We found increased EGF-R mRNA expression in 6 of 10
dyshormonogenetic tissues when compared to normal spec-
imens. It is interesting to note that there is a significant and
positive correlation between the EGF-R mRNA expression
and EGF mRNA expression in these tissues. Other studies
have indicated high binding levels of EGF and its receptor
in hyperplasic thyroid tissues as well as in follicular and pap-
illary carcinoma (29). Higher levels were found in more ag-
gressive tumors such as anaplastic thyroid tumor. It has also
been suggested that quantification of EGF-R expression
would have a prognostic value in establishing prospective
thyroid tumor recurrence risk (22).
The expression of c-
myc
was also analyzed in our study
and was positive in all tissues analyzed. The results, how-
ever, showed no significant difference between the expres-
sion of c-
in normal specimens and dyshormonogenetic
myc
tissues. It is noteworthy to mention that the Spearman re-
gression analysis between mRNA expression of EGF, EGF-
mRNA expression of epidermal growth factor
FIG. 2.
(EGF) and epidermal growth factor receptor (EGF-R) in nor-
mal individuals (N1, N6) and patients with dyshormono-
genetic goiter (TPO [P2-P5, P7, P9]) and Tg [(P8, P10-P12])
(arbitrary optical densitometry units [AODU]).
R, and c
-myc
showed a significant negative correlation be-
tween these factors (data not shown). This may suggest a
modulation of this proto-oncogene in the cellular growth
pathways activated by EGF and its receptor. Recent studies
(27) had shown that c-
myc
proto-oncogene encodes nuclear
in sensitive cell culture systems TSH stimulates functional
processes such as iodide efflux and organification, EGF can
acutely inhibit these processes. TSH, however, can modulate
the mitogenic effect induced by EGF probably via an in-
creased presentation of EGF-R to the basolateral cell surface.
Possibly, TSH might induce a trophic effect on the thyroid
proteins that act like transcription factors and has an in-
creased expression in some subtypes of thyroid malignancy.
Moreover, its expression is positively correlated to thyroid
cancer agressiveness.
The expression of c-
and c-
has been studied by
fos
myc
Northern analysis (36). As in other types of cells, EGF first
enhance c- followed by increased c- mRNA concen-
gland
by increasing the responsiveness of the follic-
in vivo
fos
myc
ular cell to circulating growth factors, such as EGF (32).
Some recent data indicates that the EGF-R system is in-
trations. On the other hand, TSH or forskolin enhances c-
myc
mRNA concentration with the same kinetics as for EGF (36).
In human thyroid tumors, p53 mutations have been de-
scribed almost exclusively associated with the anaplastic his-
totype, but are only rarely detected in differentiated follicu-
lar and papilary carcinoma (30). Thus, we have also analyzed
the expression of tumor suppressor gene p53 in normal and
dyshormonogenetic specimens. This gene was weakly ex-
pressed in all tissues but there was no significant gene p53
expression difference between normal and dyshormono-
genetic tissues.
Expression of TGF- was detected in all tissues but there
b
was no significant difference between normal and
dyshormonogenetic tissues. It was not possible to establish
significant statistical correlation between mRNA expression
of this factor and the other growth factors and oncogenes
studied.
In conclusion, we have observed overexpression of both
EGF mRNA and EGF-R mRNA in about two-thirds of the
examined dyshormonogenetic goiter tissues. There was no
Spearman regression analysis between mRNA ex-
FIG. 3.
pression of epidermal growth factor (EGF) and epidermal
growth factor receptor (EGF-R) in normal individuals and
patients presenting dyshormonogenetic goiter (arbitrary op-
tical densitometry units [AODU]).
difference in c-myc, p53, and TGF- mRNAs between nor-
b

19
GROWTH FACTORS IN DYSHORMONOGENETIC GOITER
mal and goitrous tissues. The overexpression of EGF and
EGF-R mRNAs may suggest that this growth factor and its
receptor would play a role in continuous cellular prolifera-
tion, contributing to goiter formation.
noma with anaplastic transformation arising from a long-
standing goiter in a patient with Pendred’s syndrome. (Sub-
mitted).
12. Pelizzo MR, Bernante P, Toniato A, Fanira A 1997 Frequency
of thyroid carcinoma in a recent series of 539 consecutive
thyroidectomies for multinodular goiter. Tumori 83:653–655.
13. Dumont JE, Maenhaut C, Pirson I, Baptist M, Roger PP 1991
Growth factors controlling the thyroid gland. Bailliere’s Clin
Endocrinol Metab 5:727–754.
14. Duh Q-Y, Grossman RF 1995 Thyroid growth factors, signal
transduction pathways and oncogenes. Endocr Surg
75:412–437.
Acknowledgments
We gratefully acknowledge the technical assistance of Ana
Elisa Billerbeck and the fruitful discussions about this study
with Daniel Giannella Neto, James Fagin and Peter Kopp.
This work was supported, in part, by a FAPESP (Research
Foundation of São Paulo) grant 96/0998-4. The expert sec-
retarial work of Maria Suzette Pott is gratefully acknowl-
edged.
15. Bidey SP 1990 Control of thyroid cell and follicle growth.
Trends End Met 1:174–189.
16. Wang JF, Kilosveski V, Schramek C, Fong GH, Becks GP,
Hill DJ 1998 Presence and possible role of vascular en-
dothelial growth factor in thyroid cell growth and function.
J Endocrinol 157:5–12.
17. Spambalg D, Sharif N, Elisei R, Gross JL, Medeiros-Neto G,
Fagin JA 1996 Structural studies of the thyrotropin receptor
and Gsa in human thyroid cancers: Low prevalence of mu-
tations predicts infrequent involvement in malignant trans-
formation. J Clin Endocrinol Metab 81:3898–3901.
Dr. Pedrinola was a Clinical and Research Fellow at
Cedars Sinai Medical Center, UCLA (Los Angeles, CA), un-
der the guidance of James Fagin, M.D. (1991–1992). This pa-
per was presented, in part, at the 72nd Annual Meeting of
the American Thyroid Association, Palm Beach, Florida,
(September to October, 1999).
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Address reprint requests to:
Geraldo Medeiros-Neto, M.D., F.A.C.P.
Thyroid Unit, Hospital das Clinicas
University of Sao Paulo Medical School
Av Eneas C Aguiar 255–8A–bl.3
05403-900 Sao Paulo, SP, Brazil
medneto@ual.com.br
E-mail: