0013-7227/01/$03.00/0
Printed in U.S.A.
The Journal of Clinical Endocrinology & Metabolism 86(11):5443–5449
Copyright © 2001 by The Endocrine Society
Variable Expression of the Transcription Factors cAMP
Response Element-Binding Protein and Inducible cAMP
Early Repressor in the Normal Adrenal Cortex and in
Adrenocortical Adenomas and Carcinomas
ALESSANDRO PERI, PAOLA LUCIANI, BARBARA CONFORTI, SILVANA BAGLIONI-PERI,
FEDERICA CIOPPI, CLARA CRESCIOLI, PIETRO FERRUZZI, STEFANIA GELMINI,
GIORGIO ARNALDI, GABRIELLA NESI, MARIO SERIO, FRANCO MANTERO, AND
MASSIMO MANNELLI
Endocrine Unit (A.P., P.L., B.C., F.C., C.C., P.F., M.S., M.M.), Medical Genetics Unit (S.B.-P.), and Clinical Biochemistry
Unit (S.G.), Department of Clinical Physiopathology, University of Florence, 50139 Florence, Italy; Department of Human
Pathology and Oncology (G.N.), University of Florence, 50139 Florence, Italy; Department of Internal Medicine (G.A.),
University of Ancona, 60131 Ancona, Italy; and Department of Medical and Surgical Sciences (F.M.), University of Padova,
35131 Padova, Italy
The molecular mechanisms leading to adrenocortical tumorigenesis have been only partially elucidated so far. Because
the pituitary hormone ACTH, via activation of the cAMP pathway, regulates both cell proliferation/differentiation and steroid synthesis in the adrenal cortex, in this study we focused
on the cAMP-dependent transcription factors cAMP responsive element modulator (CREM) and cAMP responsive element binding protein (CREB). We studied CREM and CREB
expression by RT-PCR in human normal adrenal cortex (n ⴝ
3), adrenocortical adenomas (n ⴝ 8), and carcinomas (n ⴝ 8).
We found transcripts corresponding to the isoforms ␣, ␤, ␥,
and ␶2 of the CREM gene in all of the normal adrenal tissues,
in the adenomas, and in seven of eight carcinomas. On the
other hand, mRNA for the inducible cAMP early repressor
isoforms, which derive from an internal promoter of CREM
gene, was detected in the normal adrenal and in seven of eight
I
N THE ADRENAL cortex, the pituitary hormone ACTH
acts as the major activator of the cAMP-dependent pathway. ACTH regulates cell differentiation, steroid synthesis,
and, to a lesser extent, cell proliferation (1). However, the
regulation of adrenocortical cells by ACTH-driven cAMP
signaling appears to be complex, because an inhibitory role
of ACTH on cell proliferation in vitro has also been observed
(2, 3). Accordingly, complex multiple molecular mechanisms
are likely to be related to adrenocortical tumorigenesis, as it
is suggested by different reports. LOH of the ACTH receptor
(ACTH-R) gene and reduced levels of mRNA (4) have been
observed, for instance, in adrenal cancer and have been associated with cellular dedifferentiation. The observation that
approximately one third of patients with MEN type 1 have
adrenocortical tumors (5) prompted investigation of the association between mutations of the MEN 1 gene and adrenal
neoplasia. LOH at 11q13 has been described in adrenal tu-
Abbreviations: ACTH-R, ACTH receptor; CBP, CREB binding protein; CREB, cAMP responsive element binding protein; CREM, cAMP
responsive element modulator; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; ICER, inducible cAMP early repressor.
adenomas, but in only three of eight carcinomas. Similarly,
CREB transcripts were readily detectable in all normal adrenals and adenomas, whereas they were not found in four of
eight adrenal carcinomas. To further characterize the carcinomas, telomerase activity and the expression of the ACTH
receptor gene were determined. Telomerase activity in the
carcinomas resulted in levels significantly higher than in the
adenomas, whereas the levels of ACTH receptor mRNA were
lower in the carcinomas. No correlation was found in the carcinomas between the levels of the ACTH receptor transcript
and the loss of expression of CREB/inducible cAMP early repressor, suggesting that this alteration is not secondary to an
upstream disregulation at the receptor level. In conclusion,
our results suggest that an alteration in cAMP signaling may
be associated with malignancies of the adrenal cortex. (J Clin
Endocrinol Metab 86: 5443–5449, 2001)
mors, although it is not completely clear whether it involves
the menin locus or not (6, 7). Furthermore, the activation of
the proto-oncogene K-ras (8), p53 mutations (9), or overexpression of IGF II and IGF-binding protein-2 (10) or epidermal growth factor receptor (11) have been observed with
variable frequency. However, the molecular mechanisms
leading to adrenocortical tumorigenesis appear to be only
partially known and are yet to be clearly elucidated.
Most of the intracellular effects of cAMP are mediated by
the activation of PKA. PKA activates, by phosphorylation at
specific serine sites, the nuclear transcription factors cAMP
response element modulator (CREM) and cAMP response
element binding protein (CREB) (12). Phosphorylated CREB
and CREM bind as dimers to palindromic cAMP response
element sequences, thus modulating the expression of
cAMP-dependent genes. A classical cAMP response element
has been observed, for instance, in the promoter of 11␤hydroxylase (CYP11B1) or aldosterone synthase (CYP11B2)
genes (13). The peculiar aspect of CREB and CREM genes
resides in the fact that they can encode different isoforms,
either activating or inhibiting gene expression, by mecha-
5443
5444
J Clin Endocrinol Metab, November 2001, 86(11):5443–5449
nisms of alternative exon splicing, alternative promoter usage, and autoregulation of promoters (12). In particular, an
internal promoter of CREM gene directs the expression of the
repressor isoforms named inducible cAMP early repressors
(ICERs) in response to cAMP activation (12). Recently, in the
human adrenocortical cancer cell line H295R, loss of expression of CREB gene and overexpression of the activator CREM
␶, compared with the normal adrenal cortex, have been observed (14). It has been hypothesized that this pattern of
expression could be linked to cellular transformation. To
better define whether cAMP-dependent transcription factors
play a role in adrenocortical tumorigenesis, we studied the
expression of CREB, CREM, and ICER in normal human
adrenal cortex (n ⫽ 3), in cortisol-secreting adrenocortical
adenomas (n ⫽ 8), and in adrenocortical carcinomas (n ⫽ 8)
by RT-PCR.
Materials and Methods
Patients
Nineteen patients were included in the study after obtaining informed consent. Three human normal adrenal glands (from patients
undergoing nephrectomy), eight adrenocortical adenomas, and eight
adrenal carcinomas were studied. The clinical characteristics of the patients are reported in Table 1. Tissue specimens, obtained at surgery,
were immediately frozen in liquid nitrogen and stored at ⫺80 C until
RNA extraction. Hematoxylin and eosin-stained sections were prepared,
reviewed, and classified according to the criteria of Weiss (15).
Peri et al. • cAMP Signaling in Adrenal Cancer
point in which the number of competitor molecules is equal to that of
the target (tumoral ACTH-R).
Telomerase assay
Telomerase activity was measured as described previously (17).
Briefly, tissue samples (⬃100 mg) were homogenized and centrifuged.
The supernatants were frozen and stored at ⫺80 C. Protein concentration
was measured by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc.,
Hercules, CA). Protein (6 ␮g ) was used for telomerase assay. RNase
(Roche Diagnostics, Monza, Italy; 0.5 ␮g) was used for each assay for 30
min at 37 C to inactivate telomerase. Each extract was assayed in 47.2
␮l reaction mixture containing 10 mm Tris-HCl (pH 8.3), 50 mm KCl, 4.5
mm MgCl2, 1 mm dNTP, 20 pm TAG-U primer, and 0.5 ␮m LT4 gene 32
protein (Roche Diagnostics). After 60 min at 30 C for telomerase-mediated extension of TAG-U primer, the reaction mixture was subjected to
PCR. A 10 min at 72 C step followed PCR cycles after the addition of a
second reaction mixture containing 20 pm CTA-R primer and 1.5 U Taq
Gold (Perkin-Elmer Corp., Norwalk, CT). We diluted 10 ␮l each PCR
product with 490 ␮l 10 mm Tris-HCl, 1 mm EDTA (pH 7.5), and then 500
␮l ultrasensitive fluorescent dye PicoGreen (Molecular Probes, Inc., Eugene, OR). Fluorescence was measured in a spectrofluorometer RF-540
(Shimadzu) using standard wavelengths. DNA concentration was calculated for each sample on a calibration curve generated by dilutions of
a control DNA (0 –100 ␮g/liter). The final DNA concentration of each
sample was determined by subtracting the DNA amount obtained in the
same specimen after RNase treatment. Telomerase activity was calculated as the mean of duplicates for each sample and expressed as nanograms of DNA per microgram of protein. A negative control, obtained
after pretreatment of the sample with RNase, was also assayed for each
specimen.
CREB, CREM, and ICER RT-PCR
ACTH receptor expression
The gene expression of the ACTH-R was evaluated by quantitative/
competitive RT-PCR, as described previously (16). Briefly, the following
primers were used: 5⬘-ACTGTCCTCGTGTGGTTTTG-3⬘ and 5⬘-AGATGAAGACCCCGAGCAG-3⬘. A nonhomologous RNA competitor was
constructed and used in RT-PCR experiments. There was a 66-bp difference between the length of the competitor and the normal ACTH-R
transcript. Five increasing amounts of competitor were mixed with fixed
amounts of tumoral RNA after RNase-free DNase digestion. The bands
corresponding to the competitor and the transcript products were resolved by gel electrophoresis. The densitometric ratios were determined
and were then plotted against the number of RNA competitor molecules
added to each RT-PCR. The number of ACTH-R mRNA copies was
extrapolated considering the value 1 of the competitor/target ratio as the
RT-PCR was performed on total RNAs (0.5 ␮g for each reaction) using
CREB-, CREM-, and ICER-specific primers. In particular, CREB primers
spanned sequences at the 5⬘- and 3⬘-end of CREB mRNA (18). CREM
primers were able to generate two different amplified products (243 and
390 bp), corresponding to the isoforms ␣, ␤, ␥ (repressors), and ␶2
(activator) and to the activators ␶, ␶␣, and ␶1, respectively, as described
previously (19). ICER primers were designed to detect ICER I and ICER
II isoforms, as described previously (20). The primers were synthesized
by Roche Diagnostics. Preliminary experiments were performed to determine the PCR cycles corresponding to the exponential phase of amplification. Thereafter, the PCR were always stopped in the exponential
phase (35 cycles). Only in those cases in which no amplified signal was
detectable did we further assess the negativity by extending the number
of cycles. Each experiment was repeated three times to confirm the
TABLE 1. Clinical features of the patients affected by adrenocortical tumors (n ⫽ 4 –19), ACTH receptor expression, and
telomerase activity
Patient
(no.)
Age
(yr)
Sex
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
56
47
57
27
62
66
56
58
28
33
45
71
69
21
50
61
F
F
F
F
F
M
F
F
F
F
F
F
F
F
M
F
Stage at
surgery
III
II
IV
IV
III
IV
II
III
Postsurgical outcome
Tumor size
(cm)
Tumor
secretion
ACTH-R
(no. of copies*)
Telomerase activity
(ng DNA/␮g protein)
Cured (5**)
Cured (6)
Cured (5)
Cured (6)
Cured (7)
Cured (5)
Cured (2)
Cured (3)
Recurrence, mitotane
Recurrence, mitotane
Metastasis, mitotane
Metastasis, mitotane
Metastasis, died
Metastasis, died
Remission
Recurrence, mitotane
4.0
2.0
4.7
2.0
5.5
4.0
4.0
1.8
22.0
5.0
15.0
8.0
6.0
12.0
9.0
8.0
C
C
C
C
C
C
None
C
A
A
None
None
A⫹M
C
None
None
6.76
6.50
5.96
6.41
6.51
6.70
6.38
6.87
4.91
5.41
5.48
5.74
5.41
5.73
5.06
5.20
n.d.
12.7
2.3
0.6
7.6
5.0
7.0
2.7
27.6
17.8
18.8
17.8
11.5
15.8
15.2
13.2
Histological diagnosis in patients 4 –11, adenoma; in patients 12–19, carcinoma. C, Cortisol; A, androgens; M, mineralcorticoids.
*, Log (copies)/100 ng RNA; **, years of follow-up; n.d., not determined.
Peri et al. • cAMP Signaling in Adrenal Cancer
J Clin Endocrinol Metab, November 2001, 86(11):5443–5449 5445
results. The quality of RNAs was assessed by performing additional
RT-PCR using primers specific for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, as described previously (21). Finally, in
each RT-PCR experiment, a no RNA reaction was added as a negative
control. RT-PCR products were subjected to agarose gel electrophoresis,
Southern blot, hybridization to CREB-, CREM-, and ICER-specific
probes and immunochemiluminescent detection, as described previously (19, 20). The membranes were exposed to x-ray films. The time of
exposure of the film was kept constant in each experiment (5 min).
Sequence analysis of ICER RT-PCR products
RT-PCR for the determination of ICER transcripts originated signals
of different length. The specificity of the putative signals corresponding
to ICER I and II isoforms was validated by additional sequence analysis,
as described previously, using 32[P]-␥ATP-labeled primers (20). Amplified products were electrophoresed on acrylamide gel (6%) in the presence of 7 m urea. Then, the gels were blotted on Whatman 3 m filters
(Whatman International Ltd., Maidstone, UK). After drying, the filters
were transferred to an x-ray cassette and exposed to x-ray films for 12 h.
Statistical analysis
Statistical comparison between groups was performed using the t
test. Differences were considered as statistically significant at the 0.05
level.
Results
Histology, ACTH receptor expression, and
telomerase activity
RT-PCR analysis of CREB transcripts revealed the presence of a specific signal in all of the samples from normal
adrenal gland and adrenal adenoma. The specificity of the
1026-bp signal, corresponding to the full-length transcript of
CREB gene, was confirmed by Southern blotting and hybridization to a CREB-specific probe. The results, as obtained
by chemiluminescent detection, are shown in Fig. 1A (no.
1–3, normal adrenal; 4 –11, adenoma). Conversely, signals
corresponding to CREB transcripts were detectable in only
four of eight adrenocortical carcinomas (no. 12–15), whereas
no amplified signal was in any case obtained (experiments
were repeated three times) in the remaining samples (no.
16 –19). In these cases, no signal was detectable, even after
extending the number of PCR cycles (data not shown). The
quality of RNAs was assessed by the analysis of GAPDH
transcripts. A GAPDH-specific signal was readily detectable
in all samples (Fig. 1B), thus excluding that the absence of
CREB transcripts in some samples was due to RNA degradation. No significant difference in ACTH-R mRNA levels
was observed between the carcinomas expressing and those
not expressing CREB (5.38 ⫾ 0.17 vs. 5.35 ⫾ 0.15 log(no.
copies)/100 ng RNA; mean ⫾ se, P ⫽ 0.88).
Analysis of CREM transcripts
Total RNA from normal adrenal glands (n ⫽ 3), adrenocortical adenomas (n ⫽ 8), and adrenocortical carcinomas
(n ⫽ 8) was subjected to RT-PCR. The histological features of
the adrenal carcinomas, according to the criteria of Weiss
(15), are shown in Table 2.
In addition to histological assessment and a prolonged
follow-up (4.9 ⫾ 0.6 yr; mean ⫾ se) of the patients with
adenoma, further characterization was performed on the
tissue samples. ACTH-R expression, which can be altered in
adrenal carcinoma (4), was determined by competitive RTPCR and was found to be significantly higher in adrenal
adenomas than in carcinomas [6.51 ⫾ 0.1 vs. 5.37 ⫾ 0.1 log(no.
copies)/100 ng RNA; mean ⫾ se; P ⫽ 0.000001] (Table 1).
Telomerase activity, which has been shown to be a potential
marker for differentiating benign from malignant adrenal
tumors (22, 23), was also determined, and it was found to be
higher in the carcinomas (adenomas, 5.41 ⫾ 1.54, vs. carcinomas, 17.21 ⫾ 1.72 ng DNA/␮g protein; mean ⫾ se; P ⫽
0.00023) (Table 1).
TABLE 2. Histological data of the adrenocortical carcinomas
(n ⫽ 12–19)
Patient
(no.)
Necrosis
Mitotic index
(no./10 hpf)
Vascular
invasion
Capsular
invasion
12
13
14
15
16
17
18
19
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
10
16
8
12
21
40
14
16
⫹
⫺
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
hpf, High-power fields.
Analysis of CREB transcripts
CREM-specific primers, spanning sequences from exon B
(sense primer) and from exon D (antisense primer) of CREM
gene, were designed for RT-PCR experiments. These primers
have been designed and used previously in our laboratory
(19) and are able to generate two specific signals corresponding to different CREM isoforms (see Materials and Methods).
The expected signal of 243 bp, corresponding to the CREM
repressors ␣, ␤, and ␥ and to the activator ␶2, was detected
in all RNAs from normal adrenals (no. 1–3), adenomas (no.
4 –11), and carcinomas (no. 12–18), with the exception of one
case (no. 19). The specificity of the signal was validated by
hybridization of RT-PCR products to a CREM-specific probe,
and the results are shown in Fig. 2A. However, in no case was
the presence of the 390-bp signal corresponding to the activators ␶, ␶␣, and ␶1, which was readily detectable in other cell
systems such as germ cells (19), observed in the adrenal
cortex.
Analysis of ICER transcripts
For the detection of ICER transcripts by RT-PCR, primers
were selected from the internal promoter and exon ␥ (sense
primer) and from exon Ib (antisense primer) of CREM gene.
These primers were designed to detect ICER I (657 bp) and
ICER II (257 bp) isoforms. Amplified products were hybridized to a specific oligonucleotide probe. Signals corresponding to ICER I and ICER II transcripts were detected in all
normal adrenal glands (Fig. 2B). In adrenocortical adenomas,
ICER I and II transcripts were observed in all samples, with
the exception of one case (no. 7). Conversely, in adrenal
carcinomas, the presence of ICER I and II mRNA was detected in only three of eight cases (no. 12–14), whereas no
detectable levels of expression were repeatedly found in the
remaining cases (no. 15–19) (Fig. 2B). In those cases in which
5446
J Clin Endocrinol Metab, November 2001, 86(11):5443–5449
Peri et al. • cAMP Signaling in Adrenal Cancer
FIG. 1. A, Chemilumigram showing the hybridization pattern of RT-PCR products corresponding to the full-length CREB transcript, using a
CREB-specific probe. B, Ethidium bromide-stained gel showing GAPDH-specific RT-PCR products. Patients are numbered as follows: 1–3,
normal adrenal cortex; 4 –11, adrenocortical adenomas; 12–19, adrenocortical carcinomas. St, DNA molecular weight marker VI (Roche
Diagnostics Italia); N, no RNA control reaction.
no signal was detected, the negativity was confirmed even
after extension of the number of PCR cycles (data not shown).
Sequence analysis of the different amplified products, which
was performed in samples from normal adrenals as well as
from adenomas and carcinomas, confirmed that the 657- and
257-bp signals correspond to ICER I and ICER II, respectively
(data not shown), as described previously in pituitary adenomas (20). The middle two amplified fragments, previously
detected also in pituitary adenomas (20), contain partial sequences of ICER I (data not shown) and might correspond to
different, so far undescribed, ICER isoforms, which will need
further characterization.
Discussion
ACTH, by activating the cAMP-dependent pathway, has
a moderate effect on adrenal cell proliferation or even a mild
anti-proliferative effect in vitro (2, 3), whereas it plays a
pivotal role in regulating steroid hormone synthesis (1) and,
hence, in maintaining a differentiated phenotype. LOH of the
ACTH receptor gene and reduced expression of ACTH receptor mRNA have been detected in a subset of adrenocortical carcinomas (4). Because LOH is a characteristic of many
tumor types, it has been suggested that the ACTH-R gene
may act as a tumor suppressor gene and that the LOH of this
gene may result in loss of differentiation and in growth
advantage. The possible involvement of the cAMP-dependent pathway in neoplastic transformation of adrenocortical
cells has been recently highlighted by the identification of the
gene for Carney complex, a disease characterized by different
clinical features, including the presence of pigmented adrenocortical tumors. In fact, mutations of the gene encoding the
protein kinase type I-␣ regulatory subunit, an apparent tumor suppressor gene, have been detected in a subset of
patients with this disease (24).
In this report, we focused on two downstream targets of
the cAMP-dependent pathway, i.e. CREM and CREB, and we
investigated their expression in the normal adrenal gland as
well as in adrenocortical adenomas and carcinomas. We detected CREM-specific transcripts corresponding to the transcriptional activator ␶2 and to the inhibitors ␣, ␤, and ␥ in
RNAs from all normal adrenals, adenomas, and carcinomas,
with only one exception in the last group. In no case were
transcripts corresponding to CREM activators ␶, ␶␣, and ␶1
detectable. Therefore, an altered pattern of CREM expression
does not appear to be a molecular feature associated with
adrenal tumorigenesis. On the other hand, different patterns
of expression of ICER isoforms, which derive from an internal promoter of the CREM gene, were observed. In particular, whereas ICER isoforms were readily detectable in all
normal adrenal glands and adenomas (with the exception of
Peri et al. • cAMP Signaling in Adrenal Cancer
J Clin Endocrinol Metab, November 2001, 86(11):5443–5449 5447
FIG. 2. A, Chemilumigram showing the pattern of hybridization of RT-PCR products corresponding to CREM transcripts (␣, ␤, ␥, and ␶2
isoforms), using a CREM-specific probe. B, Chemilumigram showing the hybridization pattern of RT-PCR products corresponding to ICER
transcripts, using an ICER-specific probe. Patients are numbered as in Fig. 1.
one case), lack of expression was found in five of eight carcinomas. Similarly, CREB transcripts were consistently observed in normal adrenal cortex and in adrenal adenomas,
whereas detectable mRNA levels were not found in half of
the adrenal cancers. The CREB transcript that we detected
corresponds to the full-length transcript of CREB gene,
which originates a transcriptional activator, upon phosphorylation at Ser-133. Conversely, CREB repressors originate by
mechanisms of alternative exon splicing or by alternative
start sites of translation of CREB gene. As a consequence, lack
of transcription of one or more exons occurs, and the resulting proteins cannot undergo phosphorylation-mediated activation. In keeping with our results, in a recent report the
absence of CREB expression was observed in the human
adrenocortical cancer cell line H295R (14). However, at variance with our findings, in that study a compensatory overexpression of CREM ␶, which is usually absent or expressed
at a very low level in the normal adrenal cortex, was detected
in H295 cells (14). This apparent discrepancy might be due
to the different cell system (i.e. adrenal tumoral tissues obtained at surgery vs. a single cultured cell line). In view of our
results, it is noteworthy that both the promoter of CREB gene
and the internal promoter of CREM gene that directs the
expression of ICER isoforms, but not the upstream promoter,
are autoregulated by cAMP. In fact, three CREs are present
in the promoter region of CREB gene (25); two pairs of closely
spaced CREs are contained in the internal promoter of CREM
gene (26). As a result, activation of the cAMP pathway, by
enhancing the levels of phosphorylated, hence activated,
CREB, stimulates CREB and ICER expression. These consid-
erations, in conjunction with our experimental data, support
the hypothesis that adrenal malignancies can be associated
to an alteration of the cAMP-dependent signaling. In this
scenario, the lack of expression of CREB (and/or ICER) in a
consistent percentage of cases of adrenal carcinoma may be
regarded as a marker of loss of cell differentiation. To clarify
whether or not the absence of CREB/ICER mRNA was a
consequence of a different molecular alteration such as reduced ACTH-R expression, which may be a feature of adrenal cancer (4), the levels of transcript of the ACTH-R gene
were determined. The expression levels of this gene did not
differ between the carcinomas expressing CREB/ICER and
those showing loss of CREB/ICER expression, thus suggesting that the absence of CREB/ICER mRNA in a subset of
adrenal carcinomas does not appear to be secondary to an
upstream disregulation at the receptor level. Interestingly,
CREB activation via Ser133-phosphorylation has been related
to cell differentiation in different tissues, such as the brain
(27) and the adipose tissue (28). In the blood system, phosphorylated CREB has been shown to induce the differentiation of megakaryocytes (29). A CREB gain-of-function
mutant, which induced high levels of constitutive Ser133phosphorylation, has been found to promote cell differentiation in vitro (30). On the other hand, mice lacking CREB
expression have been generated; the mutant mice invariably
die from respiratory distress, associated to surfactant deficiency, immediately after birth (31). Significantly, a severe
impairment in brain and T cell development has been observed. Furthermore, transgenic mice, in which expression of
a dominant negative CREB isoform was targeted to the thy-
5448 J Clin Endocrinol Metab, November 2001, 86(11):5443–5449
roid gland, exhibited severe growth retardation and primary
hypothyroidism; histologically, the thyroid glands were
characterized by poorly developed follicles (32).
CREB-mediated activation of transcription is a multifactorial process that includes the involvement of different coactivators interacting with the transcriptional apparatus.
CREB binding protein (CBP) and p300 are two factors connecting with CREB only in its phosphorylated form (33, 34),
thus participating in the molecular events resulting in the
stimulation of gene expression. CBP and p300 take part in a
variety of cellular processes, such as cell growth, differentiation, and apoptosis (35). It is noteworthy that alterations of
the human CBP gene have been implicated in hematological
malignancies, such as acute or chronic myeloid leukemia (36,
37). Similarly, inactivation of p300 gene has been related to
leukemia (38) as well as to gastric and colorectal carcinomas
(39), suggesting that these coactivators of CREB function may
serve as tumor suppressor proteins. These data confirm that
cAMP signaling is strongly involved in the control of cell
growth and differentiation and that alterations disrupting
the integrity of this pathway may result in neoplastic
proliferation.
Because the number of cases of adrenocortical cancer that
we examined so far is limited, it is not possible to conclusively establish whether there is a relationship between the
presence or the absence of expression of CREB or ICER and
the clinical features of the patients. In a recent report we
observed that the high levels of telomerase activity in adrenal
carcinomas were positively correlated with the tumor size
(23). However, in the present study it may be noteworthy to
observe that, among the eight patients with adrenal carcinoma, the two patients who died from metastatic disease did
not show detectable levels of CREB and ICER expression.
Therefore, although any conclusive statement will be possible only after extending the number of cases examined, preliminary observations seem to suggest that the lack of expression of CREB/ICER in cases of adrenal cancer might be
linked to a more severe outcome.
In conclusion, in the present study we have demonstrated
for the first time that, whereas cAMP-dependent transcription factors are consistently expressed in the normal adrenal
gland and in adrenocortical adenomas, loss of expression of
CREB and/or ICER may be a feature of adrenal cancer.
Because cAMP signaling is involved in the processes leading
to cell differentiation, it can be hypothesized that an alteration of the cAMP-dependent transcription factor machinery
may be associated with impaired cell differentiation and with
a transformed phenotype in the adrenal cortex.
Acknowledgments
We thank Dr. Marina Scarpelli (Department of Pathology, University
of Ancona, Italy); Prof. Andrea Amorosi (Department of Pathology,
University of Catanzaro, Italy); Prof. Marco Carini (Urology Unit, Ospedale Santa Maria Annunziata, Florence, Italy); and Prof. Domenico
Borrelli and Dr. Andrea Valeri (General Surgery Unit, Ospedale Careggi,
Florence, Italy) for valuable contributions.
Received December 19, 2000. Accepted August 13, 2001.
Address all correspondence and requests for reprints to: Dr. Massimo
Mannelli, Department of Clinical Physiopathology, Endocrine Unit, Uni-
Peri et al. • cAMP Signaling in Adrenal Cancer
versity of Florence, Viale Pieraccini, 6, 50139 Florence, Italy. E-mail:
[email protected].
References
1. Simpson ER, Waterman MR 1988 Regulation of the synthesis of steroidogenic
enzymes in adrenal cortical cells by ACTH. Annu Rev Physiol 50:427– 440
2. Ramachandran J, Suyama AT 1975 Inhibition of replication of normal adrenocortical cells in culture by adrenocorticotropin. Proc Natl Acad Sci USA
72:113–117
3. Rainey WE, Hornsby PJ, Shay JW 1983 Morphological correlates of adrenocorticotropin-stimulated steroidogenesis in cultured adrenocortical cells: differences between bovine and human adrenal cells. Endocrinology 113:48 –54
4. Reincke M, Mora P, Beuschlein F, Arlt W, Chrousos GP, Allolio B 1997
Deletion of the adrenocorticotropin receptor gene in human adrenocortical
tumors: implications for tumorigenesis. J Clin Endocrinol Metab 82:3054 –3058
5. Skogseid B, Rastad J, Gobl A, Larsson C, Backlin K, Juhlin C, Akerstrom G,
Oberg K 1995 Adrenal lesion in multiple endocrine neoplasia type 1. Surgery
118:1077–1082
6. Heppner C, Reincke M, Agarwal SK, Mora P, Allolio B, Burns AL, Spiegel
AM, Marx SJ 1999 MEN1 gene analysis in sporadic adrenocortical neoplasms.
J Clin Endocrinol Metab 84:216 –219
7. Kjellman M, Roshani L, Teh BT, Kallioniemi OP, Hoog A, Gray S, Farnebo
LO, Holst M, Backdahl M, Larsson C 1999 Genotyping of adrenocortical
tumors: very frequent deletions of the MEN 1 locus in 11q13 and of a 1centimorgan region in 2p16. J Clin Endocrinol Metab 84:730 –735
8. Lin SR, Hsu CH, Tsai JH, Wang JY, Hsieh TJ, Wu CH 2000 Decreased GTPase
activity of K-ras mutants deriving from human functional adrenocortical tumours. Br J Cancer 82:1035–1040
9. Reincke M, Karl M, Travis WH, Mastorakos G, Allolio B, Linehan HM,
Chrousos GP 1994 p53 mutations in human adrenocortical neoplasms: immunohistochemical and molecular studies. J Clin Endocrinol Metab 78:
790 –794
10. Boulle N, Logié A, Gicquel C, Perin L, Le Bouc Y 1998 Increased levels of
insulin-like growth factor II (IGF-II) and IGF-binding protein-2 are associated
with malignancy in sporadic adrenocortical tumors. J Clin Endocrinol Metab
83:1713–1720
11. Kamio T, Shigematsu K, Sou H, Kawai K, Tsuchiyama H 1990 Immunohistochemical expression of epidermal growth factor receptors in human adrenocortical carcinoma. Hum Pathol 21:277–282
12. De Cesare D, Sassone-Corsi P 2000 Transcriptional regulation by cAMPresponsive factors. Prog Nucleic Acid Res Mol Biol 64:343–369
13. Clyne CD, Zhang Y, Slutsker L, Mathis JM, White PC, Rainey WE 1997
Angiotensin II and potassium regulate human CYP11B2 transcription through
common cis-elements. Mol Endocrinol 11:638 – 649
14. Groussin L, Massais JF, Bertagna X, Bertherat J 2000 Loss of expression of the
ubiquitous transcription factor cAMP responsive element- binding protein
(CREB) and compensatory overexpression of the activator CREM t in the
human adrenocortical cancer cell line H295R. J Clin Endocrinol Metab 85:
345–354
15. Weiss LM 1984 Comparative histologic study of 43 metastasizing and nonmetastasizing adrenal cortical tumors. Am J Surg Pathol 8:163–169
16. Arnaldi G, Mancini V, Costantini C, Giovagnetti M, Petrelli M, Masini A,
Bertagna X, Mantero F 1998 ACTH receptor mRNA in human adrenocortical
tumors: overexpression in aldosteronomas. Endocr Res 24:845– 849
17. Gelmini S, Caldini A, Becherini L, Capaccioli S, Pazzagli M, Orlando C 1998
Rapid, quantitative nonisotopic assay for telomerase activity in human tumors.
Clin Chem 44:2133–2138
18. Berkowitz LA, Gilman MZ 1990 Two distinct forms of active transcription
factor CREB (cAMP response element binding protein). Proc Natl Acad Sci
USA 87:5258 –5262
19. Peri A, Krausz C, Cioppi F, Granchi S, Forti G, Francavilla S, Serio M 1998
Cyclic adenosine 3⬘,5⬘-monophosphate-responsive element modulator gene
expression in germ cells of normo- and oligoazoospermic men. J Clin Endocrinol Metab 83:3722–3726
20. Peri A, Conforti B, Baglioni-Peri S, Luciani P, Cioppi F, Buci L, Corbetta S,
Ballaré E, Serio M, Spada A 2001 Expression of cyclic adenosine 3⬘,5⬘-monophosphate (cAMP)-responsive element binding protein and inducible-cAMP
early repressor genes in growth hormone-secreting pituitari adenomas with or
without mutations of the Gs␣ gene. J Clin Endocrinol Metab 86:2111–2117
21. Dveksler GS, Basile AA, Dieffenbach CW 1992 Analysis of gene expression:
use of oligonucleotide primers for glyceraldehyde-3-phosphate dehydrogenase. PCR Methods Appl 1:283–285
22. Hirano Y, Fujita K, Suzuki K, Ushiyama T, Ohtawara Y, Tsuda F 1998
Telomerase activity as an indicator of potentially malignant adrenal tumors.
Cancer 83:772–776
23. Mannelli M, Gelmini S, Arnaldi G, Becherini L, Bemporad D, Crescioli C,
Pazzagli M, Mantero F, Serio M, Orlando C 2000 Telomerase activity is
significantly enhanced in malignant adrenocortical tumors in comparison to
benign adrenocortical adenomas. J Clin Endocrinol Metab 85:468 – 470
24. Kirschner LS, Carney JA, Pack SD, Taymans SE, Giatzakis C, Cho YS,
Peri et al. • cAMP Signaling in Adrenal Cancer
25.
26.
27.
28.
29.
30.
31.
32.
Cho-Chung YS, Stratakis CA 2000 Mutations of the gene encoding the protein
kinase A type I-alpha regulatory subunit in patients with the Carney complex.
Nat Genet 26:89 –92
Meyer TE, Waeber G, Lin J, Beckmann W, Habener JF 1993 The promoter of
the gene encoding 3⬘,5⬘-cyclic adenosine monophosphate (cAMP) response
element-binding protein contains cAMP response elements: evidence for positive autoregulation of gene transcription. Endocrinology 132:770 –780
Molina CA, Foulkes NS, Lalli E, Sassone-Corsi P 1993 Inducibility and
negative autoregulation of CREM: an alternative promoter directs the expression of ICER, an early response repressor. Cell 75:875– 886
Reddy YR, Basu A, Bannerman P, Ikegaki N, Reddy CD, Pleasure D 1999
ZPK inhibits PKA induced transcriptional activation by CREB and blocks
retinoic acid induced neuronal differentiation. Oncogene 18:4474 – 4484
Reusch JE, Colton LA, Klemm DJ 2000 CREB activation induces adipogenesis
in 3T3–L1 cells. Mol Cell Biol 20:1008 –1020
Zauli G, Gibellini D, Vitale M, Secchiero P, Celeghini C, Bassini A, Pierpaoli
S, Marchisio M, Guidotti L, Capitani S 1998 The induction of megakaryocyte
differentiation is accompanied by selective Ser133 phosphorylation of the transcription factor CREB in both HEL cell line and primary CD34⫹ cells. Blood
92:472– 480
Du K, Asahara H, Jhala US, Wagner BL, Montminy M 2000 Characterization
of a CREB gain-of-function mutant with constitutive transcriptional activity in
vivo. Mol Cell Biol 20:4320 – 4327
Rudolph D, Tafuri A, Gass P, Haemmerling GJ, Arnold B, Schuetz G 1998
Impaired fetal T cell development and perinatal lethality in mice lacking the
cAMP response element binding protein. Proc Natl Acad Sci USA 95:4481–
4486
Nguyen LQ, Kopp P, Martinson F, Stanfield K, Roth SI, Jameson JL 2000 A
J Clin Endocrinol Metab, November 2001, 86(11):5443–5449 5449
33.
34.
35.
36.
37.
38.
39.
dominant negative CREB (camp response element-binding protein) isoform
inhibits thyrocyte growth, thyroid-specific gene expression, differentiation,
and function. Mol Endocrinol 14:1448 –1461
Chrivia JC, Kwok RP, Lamb N, Hagiwara M, Montminy MR, Goodman RH
1993 Phosphorylated CREB binds specifically to the nuclear protein CBP.
Nature 365:855– 859
Arany Z, Sellers WR, Livingston DM, Eckner R 1994 E1A-associated p300 and
CREB-associated CBP belong to a conserved family of coactivators. Cell 7:
799 – 800
Shikama N, Lee CW, France S, Delavaine L, Lyon J, Krstic-Demonacos M,
La Thangue NB 1999 A novel cofactor for p300 that regulates p53 response.
Mol Cell 4:365–376
Borrow J, Stanton Jr VP, Andresen JM, Becher R, Behm FG, Chaganti RS,
Civin CI, Disteche C, Dube I, Frischauf AM, Horsman D, Mitelman F,
Volinia S, Watmore AE, Housman DE 1996 The translocation t(8;16)(p11;p13)
of acute myeloid leukaemia fuses a putative acetyltransferase to the CREBbinding protein. Nat Genet 14:33– 41
Rowley JD, Reshmi S, Sobulo O, Musvee T, Anastasi J, Raimondi S, Schneider NR, Barredo JC, Cantu ES, Schlegelberger B, Behm F, Doggett NA,
Borrow J, Zeleznik-Le N 1997 All patients with the T(1;16)(q23;p13.3) that
involves MLL and CBP have treatment-related hematologic disorders. Blood
90:535–541
Ida K, Kitabayashi I, Taki T, Taniwaki M, Noro K, Yamamoto M, Ohki M,
Hayashi Y 1997 Adenoviral E1A-associated protein p300 is involved in acute
myeloid leukemia with t(1;22)(q23;q13). Blood 90:4699 – 4704
Muraoka M, Konishi M, Kikuchi-Yanoshita R, Tanaka K, Shitara N, Chong
JM, Iwama T, Miyaki M 1996 p300 gene alterations in colorectal and gastric
carcinomas. Oncogene 12:1565–1569